RSV-specific binding molecules and means for producing them

ABSTRACT

The invention provides antibodies and functional equivalents thereof which are capable of specifically binding RSV, and means and methods for producing them.

This application is a divisional application of U.S. patent applicationSer. No. 12/600,950, now U.S. Pat. No. 8,562,996, issued Oct. 22, 2013,which is the §371 U.S. national phase application of InternationalPatent Application No. PCT/NL2008/050333, filed on May 30, 2008, whichclaims priority to European Patent Application No. 07109472.6, filedJun. 1, 2007, all of which are incorporated herein by reference.

The invention relates to the fields of biology and medicine.

Respiratory Syncytial Virus (RSV) is a common cold virus belonging tothe family of paramyxovirus. RSV is virulent, easily transmissible andthe most common cause of lower respiratory tract disease in children ofless than 2 years of age. Up to 98% of children attending day care willbe infected in a single RSV season. Between 0.5% and 3.2% of childrenwith RSV infection require hospitalization. Approximately 90,000hospital admissions and 4500 deaths per year were reported in UnitedStates. Major risk factors for hospitalization due to RSV are prematurebirth, chronic lung disease, congenital heart disease, compromisedimmunity, and age younger than 6 weeks in otherwise healthy children. Noeffective treatment of RSV positive bronchiolitis beside supportive carein the form of adequate nutrition and oxygen therapy is available.Antiviral therapies such as Ribavirin have not been proven to beeffective in RSV infection. One monoclonal antibody, Palivizumab (alsocalled Synagis), is registered for prophylaxis against RSV infection.Palivizumab is a genetically engineered (humanized) monoclonal antibodyto the fusion protein of RSV. However, Palivizumab is not alwayseffective. Therefore, there is a need in the art for alternativeantibodies and therapies against RSV.

It is an object of the present invention to provide means and methodsfor counteracting and/or preventing an RSV-related disease. It is afurther object of the invention to provide alternative and/or improvedantibodies against RSV, or functional equivalents of such antibodies,and to provide stable cells capable of producing antibodies—orfunctional equivalents thereof—against RSV.

The present invention provides antibodies and functional equivalentsthereof which are capable of specifically binding RSV. Such antibodiesand/or functional equivalents, also called herein “anti-RSV antibodies”or “RSV-specific antibodies”, are capable of specifically binding atleast one component of RSV, such as for instance an epitope of an RSVprotein, Non-specific sticking is not encompassed by the term“specifically binding”. Anti-RSV antibodies and functional equivalentsaccording to the present invention are particularly suitable forcounteracting and/or at least in part preventing an RSV-infection and/oradverse effects of an RSV infection. One particularly preferred anti-RSVantibody according to the present invention is the antibody designated“D25”, which has a heavy chain region and a light chain region asdepicted in FIGS. 11A-C. The CDR sequences of D25, which in particularcontribute to the antigen-binding properties of D25, are depicted inFIGS. 11B and 11C. Antibody D25 appears to have superior characteristicsas compared to the registered anti-RSV antibody Palivizumab (FIG. 8).For instance, D25 has an IC50 value of about 0.4-1.5 ng/ml in an invitro neutralization assay wherein HEp-2 cells are infected with RSV,whereas Palivizumab has an IC50 value of about 453 ng/ml.

A functional equivalent of an antibody is defined herein as a functionalpart, derivative or analogue of an antibody.

A functional part of an antibody is defined as a part which has at leastone same property as said antibody in kind, not necessarily in amount.Said functional part is capable of binding the same antigen as saidantibody, albeit not necessarily to the same extent. A functional partof an antibody preferably comprises a single domain antibody, a singlechain antibody, a single chain variable fragment (scFv), a Fab fragmentor a F(ab′)₂ fragment.

A functional derivative of an antibody is defined as an antibody whichhas been altered such that at least one property—preferably anantigen-binding property—of the resulting compound is essentially thesame in kind, not necessarily in amount. A derivative is provided inmany ways, for instance through conservative amino acid substitution,whereby an amino acid residue is substituted by another residue withgenerally similar properties (size, hydrophobicity, etc), such that theoverall functioning is likely not to be seriously affected.

A person skilled in the art is well able to generate analogous compoundsof an antibody. This is for instance done through screening of a peptidelibrary or phage display library. Such an analogue has essentially atleast one same property as said antibody in kind, not necessarily inamount.

As is well known by the skilled person, a heavy chain of an antibody isthe larger of the two types of chains making up an immunoglobulinmolecule. A heavy chain comprises constant domains and a variabledomain, which variable domain is involved in antigen binding. A lightchain of an antibody is the smaller of the two types of chains making upan immunoglobulin molecule. A light chain comprises a constant domainand a variable domain. The variable domain is, together with thevariable domain of the heavy chain, involved in antigen binding.

Complementary-determining regions (CDRs) are the hypervariable regionspresent in heavy chain variable domains and light chain variabledomains. The CDRs of a heavy chain and the connected light chain of anantibody together form the antigen-binding site.

Now that the present invention provides the insight that the CDRsequences depicted in FIG. 11 provide desired RSV-bindingcharacteristics, a skilled person is well capable of generating variantscomprising at least one altered CDR sequence. For instance, conservativeamino acid substitution is applied. Conservative amino acid substitutioninvolves substitution of one amino acid with another with generallysimilar properties (size, hydrophobicity, etc), such that the overallfunctioning is likely not to be seriously affected,

It is also possible to change at least one CDR sequence depicted in FIG.11 in order to generate a variant antibody, or a functional equivalentthereof, with at least one altered property as compared to D25.Preferably, an antibody or functional equivalent is provided comprisinga CDR sequence which is at least 70% identical to a CDR sequence asdepicted in FIG. 11, so that the favorable binding characteristics ofD25 are at least in part maintained or even improved. A CDR sequence asdepicted in FIG. 11 is preferably altered such that the resultingantibody or functional equivalent comprises at least one improvedproperty, such as for instance an improved binding affinity, selectivityand/or stability, as compared to D25. Variant antibodies or functionalequivalents thereof comprising an amino acid sequence which is at least70% identical to a CDR sequence as depicted in FIG. 11 are thereforewithin the scope of the present invention. Various methods are availablein the art for altering an amino acid sequence. For instance, a heavychain or light chain sequence with a desired CDR sequence isartificially synthesized. Preferably, a nucleic acid sequence encoding aCDR sequence is mutated, for instance using random- orsite-directed-mutagenesis.

In a first aspect the invention thus provides an isolated, synthetic orrecombinant antibody or a functional equivalent thereof which is capableof specifically binding Respiratory Syncytial Virus and which comprises:

a heavy chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence NYIIN (SEQ ID NO: 1), and/or

a heavy chain CDR2 sequence comprising a sequence which is at least 75%identical to the sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2), and/or

a heavy chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3), and/or

a light chain CDR1 sequence comprising a sequence which is at least 85%identical to the sequence QASQDIVNYLN (SEQ ID NO: 4), and/or

a light chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence VASNLET (SEQ ID NO: 5).

Preferably, said antibody also comprises a light chain CDR3 sequencecomprising a sequence which is at least 70% identical to the sequenceQQYDNLP (SEQ ID NO: 6).

Preferably, an antibody or a functional equivalent according to theinvention comprises a CDR sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90% identical to at least one of the CDR sequences depicted inFIG. 11A. Most preferably, an antibody or a functional equivalentaccording to the invention comprises a CDR sequence which is at least95% identical to at least one of the CDR sequences depicted in FIG. 11A.The particularly preferred antibody D25, described above, comprises CDRsequences which consist of the CDR sequences depicted in FIG. 11A. Aparticularly preferred embodiment according to the invention thusprovides an isolated, synthetic or recombinant antibody or a functionalequivalent thereof which is capable of specifically binding RespiratorySyncytial Virus and which comprises:

a heavy chain CDR1 sequence comprising the sequence NYIIN (SEQ ID NO:1), and/or

a heavy chain CDR2 sequence comprising the sequence GIIPVLGTVHYAPKFQG(SEQ ID NO: 2), and/or

a heavy chain CDR3 sequence comprising the sequence ETALVVSTTYLPHYFDN(SEQ ID NO: 3), and/or a light chain CDR1 sequence comprising thesequence QASQDIVNYLN (SEQ ID NO: 4), and/or

a light chain CDR2 sequence comprising the sequence VASNLET (SEQ ID NO:5).

Preferably, said antibody also comprises a light chain CDR3 sequencecomprising the sequence QQYDNLP (SEQ ID NO: 6).

In one embodiment an antibody or functional equivalent is provided whichcomprises the three heavy chain CDR sequences and the three light chainCDR sequences as depicted in FIGS. 11B and 11C, or sequences that are atleast 70%, preferably at least 80%, more preferably at least 85%identical thereto. Further provided is therefore an isolated, syntheticor recombinant antibody or a functional equivalent thereof whichcomprises a heavy chain CDR1 sequence comprising a sequence which is atleast 70% identical to the sequence NYIIN (SEQ ID NO: 1) and a heavychain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) and a heavychain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3) and a lightchain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence QASQDIVNYLN (SEQ ID NO: 4) and a light chainCDR2 sequence comprising a sequence which is at least 70% identical tothe sequence VASNLET (SEQ ID NO: 5), and a light chain CDR3 sequencecomprising a sequence which is at least 70% identical to the sequenceQQYDNLP (SEQ ID NO: 6). Said antibody or functional equivalentpreferably comprises CDR sequences which are at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainCDR sequences and the light chain CDR sequences as depicted in FIGS. 11Band 11C. An antibody or functional equivalent comprising the abovementioned heavy chain CDR1, CDR2 and CDR3 sequences as well as the abovementioned light chain CDR1, CDR2 and CDR3 sequences is also provided.

Antibodies or functional equivalents thereof comprising a variable heavychain amino acid sequence which is at least 70% identical to the heavychain sequence as depicted in FIG. 11 is also provided. Such heavy chainsequences provide desired RSV-binding properties, as evidenced byantibody D25. Further provided is therefore an antibody or a functionalequivalent thereof, having a heavy chain sequence comprising a sequencewhich is at least 70% identical to the sequence

(SEQ ID NO: 7) QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSS.Moreover, variable light chain amino acid sequences which are at least70% identical to the light chain sequence as depicted in FIG. 11 alsoprovide desired RSV-binding properties, as evidenced by antibody D25. Anantibody, or a functional equivalent thereof having a light chainsequence which is at least 70% identical to the sequence

(SEQ ID NO: 8) DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGT KVEIKRTVis therefore also provided. An antibody or functional part according tothe invention preferably comprises a variable heavy chain sequenceand/or a variable light chain sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and/or the light chain sequence as depicted in FIG. 11. Thehigher the homology, the more closely said antibody or functional partresembles antibody D25. An antibody or functional part according to theinvention preferably comprises a heavy chain as well as a light chainwhich resemble the heavy and light chain of D25. Further provided istherefore an antibody or functional part comprising a heavy chainsequence and a light chain sequence which are at least 70%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and the light chain sequence as depicted in FIG. 11.

One embodiment provides an antibody or functional equivalent thereofcomprising a heavy chain sequence consisting of the heavy chain sequenceas depicted in FIG. 11, and a light chain sequence consisting of thelight chain sequence as depicted in FIG. 11. Alternatively, as is wellknown by the skilled person, it is possible to generate a shortenedheavy chain or light chain sequence while maintaining a binding propertyof interest. Preferably, such a shortened heavy chain or light chain isgenerated which has a shorter constant region, as compared to theoriginal heavy or light chain. The variable domain is preferablymaintained. For instance, a Fab fragment or F(ab)₂ fragment based on aheavy chain sequence or light chain sequence depicted in FIG. 11 isproduced. A functional equivalent of an antibody comprising at least afunctional part of a sequence as depicted in FIG. 11 is therefore alsoprovided. Said functional part has a length of at least 20 amino acidsand comprises a sequence which is at least 70% identical to the heavychain CDR1 sequence depicted in FIGS. 11B and 11C, and/or a sequencewhich is at least 75% identical to the heavy chain CDR2 sequencedepicted in FIGS. 11B and 11C, and/or a sequence which is at least 70%identical to the heavy chain CDR3 sequence depicted in FIGS. 11B and11C, and/or a sequence which is at least 85% identical to the lightchain CDR1 sequence depicted in FIGS. 11B and 11C, and/or a sequencewhich is at least 70% identical to the light chain CDR2 sequencedepicted in FIGS. 11B and 11C. Preferably, said functional part alsocomprises a sequence which is at least 70% identical to the light chainCDR3 sequence depicted in FIGS. 11B and 11C.

Another particularly preferred anti-RSV antibody according to thepresent invention is the antibody designated “AM14”, which has a heavychain region and a light chain region as depicted in FIG. 14A. The CDRsequences of AM14, which in particular contribute to the antigen-bindingproperties of AM14, are also depicted in FIG. 14A.

Now that the present invention provides the insight that the CDRsequences depicted in FIG. 14A provide desired RSV-bindingcharacteristics, a skilled person is well capable of generating variantscomprising at least one altered CDR sequence. For instance, conservativeamino acid substitution is applied. Conservative amino acid substitutioninvolves substitution of one amino acid with another with generallysimilar properties (size, hydrophobicity, etc), such that the overallfunctioning is likely not to be seriously affected.

It is also possible to change at least one CDR sequence depicted in FIG.14A in order to generate a variant antibody, or a functional equivalentthereof, with at least one altered property as compared to AM14.Preferably, an antibody or functional equivalent is provided comprisinga CDR sequence which is at least 70% identical to a CDR sequence asdepicted in FIG. 14A, so that the favorable binding characteristics ofAM14 are at least in part maintained or even improved. A CDR sequence asdepicted in FIG. 14A is preferably altered such that the resultingantibody or functional equivalent comprises at least one improvedproperty, such as for instance an improved binding affinity, selectivityand/or stability, as compared to AM14. Variant antibodies or functionalequivalents thereof comprising an amino acid sequence which is at least70% identical to a CDR sequence as depicted in FIG. 14A are thereforewithin the scope of the present invention. Various methods are availablein the art for altering an amino acid sequence. For instance, a heavychain or light chain sequence with a desired CDR sequence isartificially synthesized. Preferably, a nucleic acid sequence encoding aCDR sequence is mutated, for instance using random- orsite-directed-mutagenesis.

In one aspect the invention thus provides an isolated, synthetic orrecombinant antibody or a functional part, derivative and/or analoguethereof which is capable of specifically binding Respiratory SyncytialVirus and which comprises:

a heavy chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence GFSFSHYA (SEQ ID NO: 73), and/or

a heavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence ISYDGENT (SEQ ID NO: 74), and/or

a heavy chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence ARDRIVDDYYYYGMDV (SEQ ID NO: 75), and/or

a light chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence QDIKKY(SEQ ID NO: 76), and/or

a light chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence DAS, and/or

a light chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence QQYDNLPPLT (SEQ ID NO; 77).

Preferably, an antibody or a functional equivalent according to theinvention comprises a CDR sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90% identical to at least one of the CDR sequences depicted inFIG. 14A. Most preferably, an antibody or a functional equivalentaccording to the invention comprises a CDR sequence which is at least95% identical to at least one of the CDR sequences depicted in FIG. 14A.The particularly preferred antibody AM14, described above, comprises CDRsequences which consist of the CDR sequences depicted in FIG. 14A. Aparticularly preferred embodiment according to the invention thusprovides an isolated, synthetic or recombinant antibody or a functionalequivalent thereof which is capable of specifically binding RespiratorySyncytial Virus and which comprises:

a heavy chain CDR1 sequence comprising the sequence GFSFSHYA (SEQ ID NO:73), and/or

a heavy chain CDR2 sequence comprising the sequence ISYDGENT (SEQ ID NO:74), and/or

a heavy chain CDR3 sequence comprising the sequence ARDRIVDDYYYYGMDV(SEQ ID NO: 75), and/or

a light chain CDR1 sequence comprising the sequence QDIKKY (SEQ ID NO:76), and/or

a light chain CDR2 sequence comprising the sequence DAS, and/or

a light chain CDR3 sequence comprising the sequence QQYDNLPPLT (SEQ IDNO: 77).

In one embodiment an antibody or functional equivalent is provided whichcomprises the three heavy chain CDR sequences and the three light chainCDR sequences as depicted in FIG. 14A, or sequences that are at least70% identical thereto. Further provided is therefore an isolated,synthetic or recombinant antibody or a functional equivalent thereofwhich comprises a heavy chain CDR1 sequence comprising a sequence whichis at least 70% identical to the sequence GFSFSHYA (SEQ ID NO: 73) and aheavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence ISYDGENT (SEQ ID NO: 74) and a heavy chainCDR3 sequence comprising a sequence which is at least 70% identical tothe sequence ARDRIVDDYYYYGMDV (SEQ ID NO: 75) and a light chain CDR1sequence comprising a sequence which is at least 70% identical to thesequence QDIKKY (SEQ ID NO 76) and a light chain CDR2 sequencecomprising a sequence which is at least 70% identical to the sequence.DAS, and a light chain CDR3 sequence comprising a sequence which is atleast 70% identical to the sequence QQYDNLPPLT (SEQ ID NO: 77). Saidantibody or functional equivalent preferably comprises CDR sequenceswhich are at least 75%, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, most preferably at least 95%identical to the heavy chain CDR sequences and the light chain CDRsequences as depicted in FIG. 14A. An antibody or functional equivalentcomprising the above mentioned heavy chain CDR1, CDR2 and CDR3 sequencesof FIG. 14A as well as the above mentioned light chain CDR1, CDR2 andCDR3 sequences of FIG. 14A is also provided.

Antibodies or functional equivalents thereof comprising a heavy chainamino acid sequence which is at least 70% identical to a heavy chainsequence as depicted in FIG. 14A is also provided. Such heavy chainsequences provide desired RSV-binding properties, as evidenced byantibody AM14. Further provided is therefore an antibody or a functionalequivalent thereof, having a heavy chain sequence comprising a sequencewhich is at least 70% identical to the sequence

(SEQ ID NO: 78) EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS.Moreover, light chain amino acid sequences which are at least 70%identical to a light chain sequence as depicted in FIG. 14A also providedesired RSV-binding properties, as evidenced by antibody AM14. Anantibody, or a functional equivalent thereof having a light chainsequence which is at least 70% identical to the sequence

(SEQ ID NO: 79) DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTVis therefore also provided. An antibody or functional part according tothe invention preferably comprises a variable heavy chain sequenceand/or a variable light chain sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to a heavy chainsequence and/or a light chain sequence as depicted in FIG. 14A. Thehigher the homology, the more closely said antibody or functional partresembles antibody AM14. An antibody or functional part according to theinvention preferably comprises a heavy chain as well as a light chainwhich resemble the heavy and light chain of AM14. Further provided istherefore an antibody or functional part comprising a heavy chainsequence and a light chain sequence which are at least 70%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and the light chain sequence as depicted in FIG. 14A.

One embodiment provides an antibody or functional equivalent thereofcomprising a heavy chain sequence consisting of the heavy chain sequenceas depicted in FIG. 14A, and a light chain sequence consisting of thelight chain sequence as depicted in FIG. 14A. Alternatively, as is wellknown by the skilled person, it is possible to generate a shortenedheavy chain or light chain sequence while maintaining a binding propertyof interest. Preferably, such a shortened heavy chain or light chain isgenerated which has a shorter constant region, as compared to theoriginal heavy or light chain. The variable domain is preferablymaintained. For instance, a Fab fragment or F(ab′)₂ fragment based on aheavy chain sequence or light chain sequence depicted in FIG. 14A isproduced. A functional equivalent of an antibody comprising at least afunctional part of a sequence as depicted in FIG. 14A is therefore alsoprovided. Said functional part has a length of at least 20 amino acidsand comprises a sequence which is at least 70% identical to at least oneof the CDR sequences depicted in FIG. 14A.

Another particularly preferred anti-RSV antibody according to thepresent invention is the antibody designated “AM16”, which has a heavychain region and a light chain region as depicted in FIG. 14B. The CDRsequences of AM16, which in particular contribute to the antigen-bindingproperties of AM16, are also depicted in FIG. 14B.

Now that the present invention provides the insight that the CDRsequences depicted in FIG. 14B provide desired RSV-bindingcharacteristics, a skilled person is well capable of generating variantscomprising at least one altered CDR sequence. For instance, conservativeamino acid substitution is applied. Conservative amino acid substitutioninvolves substitution of one amino acid with another with generallysimilar properties (size, hydrophobicity, etc), such that the overallfunctioning is likely not to be seriously affected.

It is also possible to change at least one CDR sequence depicted in FIG.14B in order to generate a variant antibody, or a functional equivalentthereof, with at least one altered property as compared to AM16.Preferably, an antibody or functional equivalent is provided comprisinga CDR sequence which is at least 70% identical to a CDR sequence asdepicted in FIG. 14B, so that the favorable binding characteristics ofAM 16 are at least in part maintained or even improved. A CDR sequenceas depicted in FIG. 14B is preferably altered such that the resultingantibody or functional equivalent comprises at least one improvedproperty, such as for instance an improved binding affinity, selectivityand/or stability, as compared to AM16. Variant antibodies or functionalequivalents thereof comprising an amino acid sequence which is at least70% identical to a CDR sequence as depicted in FIG. 14B are thereforewithin the scope of the present invention. Various methods are availablein the art for altering an amino acid sequence. For instance, a heavychain or light chain sequence with a desired CDR sequence isartificially synthesized. Preferably, a nucleic acid sequence encoding aCDR sequence is mutated, for instance using random- orsite-directed-mutagenesis.

In one aspect the invention thus provides an isolated, synthetic orrecombinant antibody or a functional part, derivative and/or analoguethereof which is capable of specifically binding Respiratory SyncytialVirus and which comprises:

a heavy chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence GFTFSSYN (SEQ ID NO: 80), and/or

a heavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence ISAGSSYI (SEQ ID NO: 81), and/or

a heavy chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence AREDYGPGNYYSPNWFDP (SEQ ID NO: 82), and/or

a light chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence SSNIGAGYD (SEQ ID NO: 83), and/or

a light chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence GNT, and/or

a light chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence HSYDRSLSG (SEQ ID NO: 84).

Preferably, an antibody or a functional equivalent according to theinvention comprises a CDR sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90% identical to at least one of the CDR sequences depicted inFIG. 14B. Most preferably, an antibody or a functional equivalentaccording to the invention comprises a CDR sequence which is at least95% identical to at least one of the CDR sequences depicted in FIG. 14B.The particularly preferred antibody AM16, described above, comprises CDRsequences which consist of the CDR sequences depicted in FIG. 14B. Aparticularly preferred embodiment according to the invention thusprovides an isolated, synthetic or recombinant antibody or a functionalequivalent thereof which is capable of specifically binding RespiratorySyncytial Virus and which comprises:

a heavy chain CDR1 sequence comprising the sequence GFTFSSYN (SEQ ID NO:80), and/or

a heavy chain CDR2 sequence comprising the sequence ISAGSSYI (SEQ ID NO:81), and/or

a heavy chain CDR3 sequence comprising the sequence AREDYGPGNYYSPNWFDP(SEQ ID NO: 82), and/or

a light chain CDR1 sequence comprising the sequence SSNIGAGYD (SEQ IDNO: 83), and/or

a light chain CDR2 sequence comprising the sequence GNT, and/or

a light chain CDR3 sequence comprising the sequence HSYDRSLSG (SEQ IDNO: 84).

In one embodiment an antibody or functional equivalent is provided whichcomprises the three heavy chain CDR sequences and the three light chainCDR sequences as depicted in FIG. 14B, or sequences that are at least70% identical thereto. Further provided is therefore an isolated,synthetic or recombinant antibody or a functional equivalent thereofwhich comprises a heavy chain CDR1 sequence comprising a sequence whichis at least 70% identical to the sequence GFTFSSYN (SEQ ID NO: 80) and aheavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence ISAGSSYI (SEQ ID NO: 81) and a heavy chainCDR3 sequence comprising a sequence which is at least 70% identical tothe sequence AREDYGPGNYYSPNWFDP (SEQ ID NO: 82) and a light chain CDR1sequence comprising a sequence which is at least 70% identical to thesequence SSNIGAGYD (SEQ ID NO: 83) and a light chain CDR2 sequencecomprising a sequence which is at least 70% identical to the sequenceGNT, and a light chain CDR3 sequence comprising a sequence which is atleast 70% identical to the sequence HSYDRSLSG (SEQ ID NO: 84). Saidantibody or functional equivalent preferably comprises CDR sequenceswhich are at least 75%, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, most preferably at least 95%identical to the above mentioned heavy chain CDR sequences and the abovementioned light chain CDR sequences as depicted in FIG. 14B. An antibodyor functional equivalent comprising the above mentioned heavy chainCDR1, CDR2 and CDR3 sequences of FIG. 14B as well as the above mentionedlight chain CDR1, CDR2 and CDR3 sequences of FIG. 14B is also provided.

Antibodies or functional equivalents thereof comprising a heavy chainamino acid sequence which is at least 70% identical to a heavy chainsequence as depicted in FIG. 14B is also provided. Such heavy chainsequences provide desired RSV-binding properties, as evidenced byantibody AM16. Further provided is therefore an antibody or a functionalequivalent thereof, having a heavy chain sequence comprising a sequencewhich is at least 70% identical to the sequence

(SEQ ID NO: 85) EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSSYIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPWGQGTLVTVSS.Moreover, light chain amino acid sequences which are at least 70%identical to a light chain sequence as depicted in FIG. 14B also providedesired RSV-binding properties, as evidenced by antibody AM16. Anantibody, or a functional equivalent thereof having a light chainsequence which is at least 70% identical to the sequence

(SEQ ID NO: 86) QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVF GGGTKLTVis therefore also provided. An antibody or functional part according tothe invention preferably comprises a variable heavy chain sequenceand/or a variable light chain sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and/or the light chain sequence as depicted in FIG. 14B. Thehigher the homology, the more closely said antibody or functional partresembles antibody AM16. An antibody or functional part according to theinvention preferably comprises a heavy chain as well as a light chainwhich resemble the heavy and light chain of AM 16. Further provided istherefore an antibody or functional part comprising a heavy chainsequence and a light chain sequence which are at least 70%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and the light chain sequence as depicted in FIG. 14B.

One embodiment provides an antibody or functional equivalent thereofcomprising a heavy chain sequence consisting of the heavy chain sequenceas depicted in FIG. 14B, and a light chain sequence consisting of thelight chain sequence as depicted in FIG. 14B. Alternatively, as is wellknown by the skilled person, it is possible to generate a shortenedheavy chain or light chain sequence while maintaining a binding propertyof interest. Preferably, such a shortened heavy chain or light chain isgenerated which has a shorter constant region, as compared to theoriginal heavy or light chain. The variable domain is preferablymaintained. For instance, a Fab fragment or F(ab′)₂ fragment based on aheavy chain sequence or light chain sequence depicted in FIG. 14B isproduced. A functional equivalent of an antibody comprising at least afunctional part of a sequence as depicted in FIG. 14B is therefore alsoprovided. Said functional part has a length of at least 20 amino acidsand comprises a sequence which is at least 70% identical to at least oneof the CDR sequences depicted in FIG. 14B,

Another particularly preferred anti-RSV antibody according to thepresent invention is the antibody designated “AM23”, which has a heavychain region and a light chain region as depicted in FIG. 14C. The CDRsequences of AM23, which in particular contribute to the antigen-bindingproperties of AM23, are also depicted in FIG. 14C.

Now that the present invention provides the insight that the CDRsequences depicted in FIG. 14C provide desired RSV-bindingcharacteristics, a skilled person is well capable of generating variantscomprising at least one altered CDR sequence. For instance, conservativeamino acid substitution is applied. Conservative amino acid substitutioninvolves substitution of one amino acid with another with generallysimilar properties (size, hydrophobicity, etc), such that the overallfunctioning is likely not to be seriously affected.

It is also possible to change at least one CDR sequence depicted in FIG.14C in order to generate a variant antibody, or a functional equivalentthereof, with at least one altered property as compared to AM23.Preferably, an antibody or functional equivalent is provided comprisinga CDR sequence which is at least 70% identical to a CDR sequence asdepicted in FIG. 14C, so that the favorable binding characteristics ofAM23 are at least in part maintained or even improved. A CDR sequence asdepicted in FIG. 14C is preferably altered such that the resultingantibody or functional equivalent comprises at least one improvedproperty, such as for instance an improved binding affinity, selectivityand/or stability, as compared to AM23. Variant antibodies or functionalequivalents thereof comprising an amino acid sequence which is at least70% identical to a CDR sequence as depicted in FIG. 14C are thereforewithin the scope of the present invention. Various methods are availablein the art for altering an amino acid sequence. For instance, a heavychain or light chain sequence with a desired CDR sequence isartificially synthesized. Preferably, a nucleic acid sequence encoding aCDR sequence is mutated, for instance using random- orsite-directed-mutagenesis.

In one aspect the invention thus provides an isolated, synthetic orrecombinant antibody or a functional part, derivative and/or analoguethereof which is capable of specifically binding Respiratory SyncytialVirus and which comprises:

a heavy chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence GFNFHNYG (SEQ ID NO: 87), and/or

a heavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence VWYDGSKK (SEQ ID NO: 88), and/or

a heavy chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence VRDKVGPTPYFDS (SEQ ID NO: 89), and/or

a light chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence NIGSET (SEQ ID NO: 90), and/or

a light chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence DDD, and/or

a light chain CDR3 sequence comprising a sequence which is at least 70%identical to the sequence QVWDRSNYHQV (SEQ ID NO: 91).

Preferably, an antibody or a functional equivalent according to theinvention comprises a CDR sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90% identical to at least one of the CDR sequences depicted inFIG. 14C. Most preferably, an antibody or a functional equivalentaccording to the invention comprises a CDR sequence which is at least95% identical to at least one of the CDR sequences depicted in FIG. 14C.The particularly preferred antibody AM23, described above, comprises CDRsequences which consist of the CDR sequences depicted in FIG. 14C. Aparticularly preferred embodiment according to the invention thusprovides an isolated, synthetic or recombinant antibody or a functionalequivalent thereof which is capable of specifically binding RespiratorySyncytial Virus and which comprises:

a heavy chain CDR1 sequence comprising the sequence GFNFHNYG (SEQ ID NO:87), and/or

a heavy chain CDR2 sequence comprising the sequence VWYDGSKK (SEQ ID NO:88), and/or

a heavy chain CDR3 sequence comprising the sequence VRDKVGPTPYFDS (SEQID NO 89), and/or

a light chain CDR1 sequence comprising the sequence NIGSET (SEQ ID NO:90), and/or

a light chain CDR2 sequence comprising the sequence DDD, and/or

a light chain CDR3 sequence comprising the sequence QVWDRSNYHQV (SEQ IDNO: 91).

In one embodiment an antibody or functional equivalent is provided whichcomprises the three heavy chain CDR sequences and the three light chainCDR sequences as depicted in FIG. 14C, or sequences that are at least70% identical thereto. Further provided is therefore an isolated,synthetic or recombinant antibody or a functional equivalent thereofwhich comprises a heavy chain CDR1 sequence comprising a sequence whichis at least 70% identical to the sequence GFNFHNYG (SEQ ID NO: 87) and aheavy chain CDR2 sequence comprising a sequence which is at least 70%identical to the sequence VWYDGSKK (SEQ ID NO: 88) and a heavy chainCDR3 sequence comprising a sequence which is at least 70% identical tothe sequence VRDKVGPTPYFDS (SEQ ID NO: 89) and a light chain CDR1sequence comprising a sequence which is at least 70% identical to thesequence NIGSET (SEQ ID NO: 90) and a light chain CDR2 sequencecomprising a sequence which is at least 70% identical to the sequenceDDD, and a light chain CDR3 sequence comprising a sequence which is atleast 70% identical to the sequence QVWDRSNYHQV (SEQ ID NO: 91). Saidantibody or functional equivalent preferably comprises CDR sequenceswhich are at least 75%, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, most preferably at least 95%identical to the above mentioned heavy chain CDR sequences and the abovementioned light chain CDR sequences as depicted in FIG. 14C. An antibodyor functional equivalent comprising the above mentioned heavy chainCDR1, CDR2 and CDR3 sequences of FIG. 14C as well as the above mentionedlight chain CDR1, CDR2 and CDR3 sequences of FIG. 14C is also provided.

Antibodies or functional equivalents thereof comprising a heavy chainamino acid sequence which is at least 70% identical to a heavy chainsequence as depicted in FIG. 14C is also provided. Such heavy chainsequences provide desired RSV-binding properties, as evidenced byantibody AM23. Further provided is therefore an antibody or a functionalequivalent thereof, having a heavy chain sequence comprising a sequencewhich is at least 70% identical to the sequence

(SEQ ID NO: 92) EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGSKKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVG PTPYFDSWGQGTLVTVSS.Moreover, light chain amino acid sequences which are at least 70%identical to a light chain sequence as depicted in FIG. 14C also providedesired RSV-binding properties, as evidenced by antibody AM23. Anantibody, or a functional equivalent thereof having a light chainsequence which is at least 70% identical to the sequence

(SEQ ID NO: 93) SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLWYDDDDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGT KLTVis therefore also provided. An antibody or functional part according tothe invention preferably comprises a variable heavy chain sequenceand/or a variable light chain sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and/or the light chain sequence as depicted in FIG. 14C. Thehigher the homology, the more closely said antibody or functional partresembles antibody AM23. An antibody or functional part according to theinvention preferably comprises a heavy chain as well as a light chainwhich resemble the heavy and light chain of AM23. Further provided istherefore an antibody or functional part comprising a heavy chainsequence and a light chain sequence which are at least 70%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and the light chain sequence as depicted in FIG. 14C.

One embodiment provides an antibody or functional equivalent thereofcomprising a heavy chain sequence consisting of the heavy chain sequenceas depicted in FIG. 14C, and a light chain sequence consisting of thelight chain sequence as depicted in FIG. 14C. Alternatively, as is wellknown by the skilled person, it is possible to generate a shortenedheavy chain or light chain sequence while maintaining a binding propertyof interest. Preferably, such a shortened heavy chain or light chain isgenerated which has a shorter constant region, as compared to theoriginal heavy or light chain. The variable domain is preferablymaintained. For instance, a Fab fragment or F(ab′)₂, fragment based on aheavy chain sequence or light chain sequence depicted in FIG. 14C isproduced. A functional equivalent of an antibody comprising at least afunctional part of a sequence as depicted in FIG. 14C is therefore alsoprovided. Said functional part has a length of at least 20 amino acidsand comprises a sequence which is at least 70% identical to at least oneof the CDR sequences depicted in FIG. 14C.

The present invention provides RSV-specific antibodies or functionalequivalents thereof having improved properties as compared to prior artantibodies. The inventors have succeeded in generating RSV-specificantibodies with low IC₅₀ values. Such antibodies have a particular highor strong affinity for RSV and are therefore particularly suitable forcounteracting and/or at least in part preventing an RSV-infection and/oradverse effects of an RSV infection. One embodiment provides an antibodywhich has an IC₅₀ value of less than 10 ng/ml in an in vitroneutralization assay wherein HEp-2 cells are infected with RSV, and afunctional equivalent of said antibody. Said antibody or functionalequivalent preferably has an IC₅₀ value of less than 5 ng/ml, morepreferably less than 2 ng/ml. The preferred antibody D25 has an IC₅₀value of about 0.5-1.5 ng/ml in the in vitro neutralization assaydescribed in the examples (see FIG. 8).

An antibody according to the invention is preferably a human antibody.The use of human antibodies for human therapy diminishes the chance ofside-effects due to an immunological reaction in a human individualagainst non-human sequences. In another preferred embodiment an antibodyor functional part, derivative or analogue according to the invention isa chimeric antibody. This way, sequences of interest, such as forinstance a binding site of interest, can be included into an antibody orfunctional equivalent according to the invention.

The invention further provides an isolated, synthetic or recombinantnucleic acid sequence, or a functional part, derivative or analoguethereof, encoding an antibody or functional equivalent according to theinvention. Such nucleic acid is for instance isolated from a B-cellwhich is capable of producing an antibody according to the invention, asoutlined in more detail below. A preferred embodiment provides a nucleicacid sequence comprising a sequence which is at least 70% homologous toat least a functional part of a nucleic acid sequence as depicted inFIG. 11, FIG. 12, FIG. 14A, FIG. 14B and/or FIG. 14B. Said nucleic acidsequence preferably comprises a sequence which is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% homologous to at least afunctional part of a nucleic acid sequence as depicted in FIG. 11, FIG.12, FIG. 14A, FIG. 14B and/or FIG. 14B. Said functional part has alength of at least 30 nucleotides, preferably at least 50 nucleotides,more preferably at least 75 nucleotides. Preferably, said functionalpart encodes at least one nucleic acid sequence as depicted in FIG. 11A,FIG. 12, FIG. 14A, FIG. 14B and/or FIG. 14B. Said sequence is preferablya CDR sequence.

An antibody or functional equivalent according to the invention isparticularly suitable for use as a medicine or prophylactic agent. Anantibody according to the invention, or a functional part, derivative oranalogue thereof, for use as a medicament and/or prophylactic agent istherefore also herewith provided. In a particularly preferred embodimentsaid antibody comprises antibody D25, AM14, AM16 and/or AM23, or afunctional part, derivative or analogue thereof. Said medicament orprophylactic agent is preferably used for counteracting or at least inpart preventing an RSV-infection or for counteracting or at least inpart preventing adverse effects of an RSV-infection. A use of anantibody, functional part, derivative or analogue according to theinvention for the preparation of a medicament and/or prophylactic agentfor at least in part treating and/or preventing a RSV-related disorderis therefore also provided, as well as a method for at least in parttreating or preventing an RSV-related disorder, the method comprisingadministering to an individual in need thereof a therapeuticallyeffective amount of an antibody or functional equivalent according tothe invention. Said antibody preferably comprises antibody D25, AM14,AM16 and/or AM23, or a functional part, derivative or analogue thereof.

In order to counteract RSV, an antibody or functional equivalentaccording to the invention is preferably administered to an individualbefore an RSV-infection has taken place. Alternatively, an antibody orfunctional equivalent according to the invention is administered when anindividual is already infected by RSV. Said antibody or functionalequivalent is preferably administered to individuals with an increasedrisk of RSV-related disorders, such as for instance children withpremature birth, individuals with chronic lung disease, congenital heartdisease and/or compromised immunity, and children with an age youngerthan 6 weeks. Also elderly people have an increased risk of RSV-relateddisorders. Antibodies or functional equivalents according to theinvention are preferably administered orally or via one or moreinjections. Dose ranges of antibodies and/or functional equivalentsaccording to the invention to be used in the therapeutic applications asdescribed herein before are designed on the basis of rising dose studiesin the clinic in clinical trials for which rigorous protocolrequirements exist. Typical doses are between 0.1 and 10 mg per kg bodyweight. For therapeutic application, antibodies or functionalequivalents according to the invention are typically combined with apharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.Examples of suitable carriers for instance comprise keyhole limpethaemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin. Manysuitable adjuvants, oil-based and water-based, are known to a personskilled in the art. In one embodiment said adjuvant comprises Specol. Inanother embodiment, said suitable carrier comprises a solution like forexample saline.

In yet another embodiment a nucleic acid encoding an antibody orfunctional part according to the invention is used. Upon administrationof such nucleic acid, antibodies or functional equivalents are producedby the host's machinery. Produced antibodies or functional equivalentsare capable of preventing and/or counteracting RSV-infection and/or theadverse effects of an RSV-infection. A nucleic acid sequence, functionalpart, derivative and/or analogue according to the invention for use as amedicament and/or prophylactic agent is therefore also herewithprovided. Said nucleic acid is preferably used for counteracting RSV.Further provided is therefore a use of a nucleic acid sequence,functional part, derivative and/or analogue according to the inventionfor the preparation of a medicament and/or prophylactic agent for atleast in part treating and/or preventing a RSV-related disorder.

By at least a functional part of a nucleic acid of the invention ismeant a part of said nucleic acid, at least 30 base pairs long,preferably at least 50 base pairs long, more preferably at least 100base pairs long, comprising at least one expression characteristic (inkind not necessarily in amount) as a nucleic acid of the invention. Saidfunctional part at least encodes an amino acid sequence comprising asequence which is at least 70% identical to a CDR sequence as depictedin FIGS. 11A-C, FIG. 14A, FIG. 14B and/or FIG. 14C.

The invention furthermore provides an isolated antibody producing cellcapable of producing an antibody, functional part, derivative oranalogue according to the invention. Possible (but not limiting) ways ofobtaining such antibody producing cells are outlined in detail in theexamples. The inventors have developed and used a new method in order toimprove the stability of RSV-specific antibody producing cells. Usingthis method, RSV-specific antibody producing cells are generated whichare stable for at least six months. An RSV-specific antibody producingcell according to the invention, which is stable for at least nineweeks, preferably for at least three months, more preferably for atleast six months is therefore also herewith provided.

The present inventors have used their insight that the stability of anRSV-specific antibody producing cell is influenced by influencing theamount of BCL6 and/or Blimp-1 expression product within said antibodyproducing cell. The amount of BCL6 and/or Blimp-1 expression product iseither directly or indirectly influenced. Preferably the amounts of bothBCL6 and Blimp-1 expression products within said antibody producing cellare regulated, since both expression products are involved in thestability of an antibody producing cell. The stability of an antibodyproducing cell is defined as the capability of said antibody producingcell to remain in a certain developmental stage (preferably after saidcell has been brought into said stage). Different developmental stagesof a cell involve at least one different characteristic of said cell.For instance, a memory B cell is known to differentiate upon stimulationinto an antibody-secreting plasma cell via a stage which someresearchers call a plasmablast. A memory B cell, a plasmablast and aplasma cell are different developmental stages of a B cell, wherein theB cell has different characteristics. A memory B cell exhibits lowproliferation and antibody secretion. A plasmablast exhibits both higherproliferation and higher antibody secretion levels as compared to amemory B cell, whereas a plasma cell secretes high antibody levels butis not capable of proliferating. With a method of the present inventorsit has become possible to regulate the replicative life span of anantibody producing cell. A replicative life span of an antibodyproducing cell is defined herein as the time span wherein a B cell andits progeny cells are capable of replicating while maintaining theircapability of producing antibody and/or developing into a cell thatproduces antibody. Preferably the replicative life span of an antibodyproducing cell is prolonged, meaning that said antibody producing cellwill not terminally differentiate—or only after a longer period ascompared to the same kind of antibody producing cells that are currentlyused—and continue to proliferate in vitro. According to the inventors itis possible to regulate the amount of BCL6 and/or Blimp-1 expressionproduct in an antibody producing cell to such extent that the antibodyproducing cell is brought into, and/or kept in, a predetermineddevelopmental state in which the cells continue to proliferate. With amethod of the inventors it has therefore become possible to increase thereplicative life span of an antibody producing cell since it is possibleto maintain a B cell in a certain developmental stage whereinreplication occurs. Reference is made to PCT/NL2006/000625, filed by thesame applicant. The present invention provides means and methods forproducing stable RSV-specific antibody producing cells.

An antibody producing cell is defined as a cell which cell is capable ofproducing and/or secreting antibody or a functional equivalent thereof,and/or which cell is capable of developing into a cell which is capableof producing and/or secreting antibody or a functional equivalentthereof. An RSV-specific antibody producing cell is defined herein as acell capable of producing and/or secreting antibodies or functionalequivalents thereof which are capable of specifically binding RSV and/ora component of RSV, such as for instance an epitope of the RSV F(fusion) protein, the RSV G (attachment) protein or RSV SH (smallhydrophobic) protein. Preferably, said RSV-specific antibody producingcell comprises a B cell and/or a B cell-derived plasma cell. A B cell iscalled herein an antibody producing cell, even when the B cell is in astage wherein antibody production is low or not present at all, such asa naïve B cell or a memory B cell, being activated or not, because suchcells are capable of developing into cells that produce antibody, suchas a plasmablast and/or plasma cell.

An RSV-specific antibody producing cell according to the inventionpreferably comprises a mammalian cell. Non-limiting examples includeantibody producing cells derived from a human individual, rodent,rabbit, llama, pig, cow, goat, horse, ape, gorilla. Preferably, saidantibody producing cell comprises a human cell, a murine cell, a rabbitcell and/or a llama cell.

BCL6 encodes a transcriptional repressor which is required for normal Bcell and T cell development and maturation and which is required for theformation of germinal centers. (Ye, 1997). BCL6 is highly expressed ingerminal center B cells whereas it is hardly expressed in plasma cells.BCL6 inhibits differentiation of activated B cells into plasma cells.The transcriptional repressor B lymphocyte induced maturation protein-1(Blimp-1) is required for development of a B cell into a plasma cell.The human variant of Blimp-1 is named Prdm1. As used herein, anyreference to Blimp-1 includes a reference to Prdm1. Blimp-1 drivesplasma cell differentiation. BCL6 and Blimp-1 repress expression of theother; thus in a natural situation when one reaches an higher expressionlevel than the other, the stage of differentiation is enforced. In thehuman body, differentiation of plasma cells from activated naïve ormemory B cells involves downregulation of BCL6 and upregulation ofBlimp-1. In germinal center cells BCL6 expression is high and Blimp-1expression is low. In resting memory cells expression of BCL6 andBlimp-1 are low. Signals that trigger differentiation cause anupregulation of Blimp-1, and this Blimp-1 counteracts the expression ofBCL6. The stage where both BCL6 and Blimp-1 are expressed is short-livedand is called a plasmablast. With progressively increasing Blimp-1levels, BCL6 expression is extinguished, resulting in a plasma cell.

In one embodiment of the present invention, an RSV-specific antibodyproducing cell is provided wherein BCL6 and Blimp-1 are co-expressed(meaning that both BCL6 and Blimp-1 are expressed in said antibodyproducing cell for at least 1 day, preferably at least one week, morepreferably at least six weeks, most preferably at least three months.Said RSV-specific antibody producing cell is capable of proliferatingwhen an appropriate signal is provided. It has been found thatco-expression of BCL6 and Blimp-1 results in an antibody producing cellwhich is capable of both proliferating and producing antibody. BCL6 andBlimp-1 are preferably co-expressed in a B cell, preferably a human Bcell. Co-expression of BCL6 and Blimp-1 in a B cell results instabilization of said B cell in a plasmablast-like stage, Plasmablasts,like plasma cells, are capable of secreting antibody. However,plasmablasts are still capable of proliferating, whereas plasma cellshave lost their capability of proliferating. Plasma cells are thereforeunsuitable for culturing antibody-producing cell lines.

One preferred embodiment provides an RSV-specific antibody producingcell comprising an exogenous nucleic acid sequence encoding BCL6 or afunctional part, derivative and/or analogue thereof. An exogenousnucleic acid is defined herein as a nucleic acid sequence which does notnaturally belong to the genome of a cell. With such exogenous nucleicacid molecule it is possible to regulate a BCL6 concentration in anantibody producing cell independently from expression of endogenousBCL6. Hence, even if expression of endogenous BCL6 is low or absent, forinstance caused by Blimp-1, an exogenous nucleic acid sequence encodingBCL6 or a functional part, derivative and/or analogue thereof is stillcapable of producing a concentration of BCL6 which is sufficient forinfluencing the stability of an antibody producing cell. Preferably,said nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof is constitutively active, so thatBCL6 expression is maintained even when endogenous BCL6 expression ofsaid cell is inhibited by an endogenous repressor such as Blimp-1. Mostpreferably, expression of said nucleic acid sequence encoding BCL6 or afunctional part, derivative and/or analogue thereof is regulated by anexogenous inducer of repressor, so that the extent of BCL6 expression isregulated at will.

Preferably, as outlined below in more detail, an RSV-specific antibodyproducing cell according to the invention comprises an exogenous nucleicacid sequence encoding Bcl-xL or a functional part, derivative and/oranalogue thereof. If Bcl-xL or a functional part, derivative and/oranalogue thereof is present, it is possible to grow plasmablasts underconditions of low cell density. Expression of said nucleic acid sequenceencoding Bcl-xL or a functional part, derivative and/or analogue thereofis preferably regulated by an exogenous inducer of repressor, so thatthe extent of Bcl-xL expression is regulated at will. A preferredembodiment therefore provides an RSV-specific antibody producing cellcomprising:

an exogenous nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof, and/or

an exogenous nucleic acid sequence encoding Bcl-xL or a functional part,derivative and/or analogue thereof. Said RSV-specific antibody producingcell preferably comprises both an exogenous nucleic acid sequenceencoding BCL6—or a functional part, derivative and/or analoguethereof—and an exogenous nucleic acid sequence encoding Bcl-xL—or afunctional part, derivative and/or analogue thereof. Preferably,expression of said nucleic acid sequence encoding BCL6, Bcl-xL or afunctional part, derivative and/or analogue of BCL6 or Bcl-xL isregulated by an activator and/or repressor that is inducible by anexogenous compound. For instance, an inducible promoter system is usedsuch as a Tet-on or Tet-off system.

A stable RSV-specific antibody producing cell according to the inventionis preferably generated by co-expressing BCL6 and Blimp-1 in anRSV-specific antibody producing cell. An RSV-specific antibody producingcell is preferably obtained from an individual who has been exposed toRSV. Methods for isolating antibody producing cells are well known inthe art. For instance, RSV-derived compounds that are marked with alabel and/or tag are incubated with a sample of an individual who hasbeen exposed to RSV, which sample comprises antibody producing cells.RSV-specific antibody producing cells that recognize the taggedRSV-derived compounds are isolated while unbound cells are washed away.The resulting RSV-specific antibody producing cells are subsequentlystabilized by co-expressing BCL6 as well as Blimp-1.

One embodiment involves first stabilizing total antibody-producing cellsfrom an RSV exposed donor and then isolating cells that recognize thetagged RSV-derived compound. In another embodiment antibody producingcells are equipped with a (fluorescent) marker downstream their B cellreceptor (BCR, membrane expressed form of the antibody) that signalswhen the antibody producing cell binds an un-tagged/unlabeled antigenvia the BCR. Antibody producing cells in which the marker is turn areselected and are subsequently stabilized by co-expressing BCL6 as wellas Blimp-1. In another embodiment, when there are no antigen-derivedcompounds available but when there are assays available to screen forunique antibodies, total/bulk antibody producing cells are stabilized byco-expressing BCL6 as well as Blimp-1 and, optionally, also Bcl-XL.According to this embodiment, cells are cultured at low densities,preferably between 10 and 100 cells per 96-well, in the presence ofL-cells (mini bulk cultures, MBC). Culture supernatants can be useddirectly in screenings assays, like ELISA, Western blot or functionalassays like ELISPOT, neutralization assays or cell migration assays.

In one embodiment MBC are selected and, to obtain monoclonal cell linesof the antibody producing cell of interest, limiting dilution culturesare preformed and, preferably 2-3 weeks later, supernatants of thosecultures are screened again in the preferred assay.

As is well known by the skilled person, many alternative methods areavailable in the art. The above mentioned embodiments are non-limiting.

Further provided is therefore a method for producing an antibodyproducing cell, which is stable for at least three months and which iscapable of producing RSV-specific antibodies or functional equivalentsthereof, the method comprising:

increasing an expression level of Blimp-1 in a cell which is capable ofproducing RSV-specific antibodies or functional equivalents thereof; and

increasing and/or maintaining a BCL6 expression level in said cell.

With a method according to the invention it has become possible toconvert an RSV-specific memory B cell into a plasmablast-like cell andto stabilize said cell, so that rapid differentiation into a plasma celldoes not occur. This is contrary to natural development of plasma cells,wherein expression of Blimp-1 in a memory B cell results in rapiddevelopment into a plasma cell, thereby inhibiting BCL6 expression sothat the resulting plasma cell hardly expresses BCL6. One embodiment ofthe present invention thus involves co-expression of both BCL6 andBlimp-1 in an RSV-specific B cell, resulting in a cell that is capableof both proliferating and producing antibody. The BCL6 expression levelin said RSV-specific B-cell is preferably brought to, and maintained at,essentially the same level or at a higher level as compared to aplasmablast. This way a stable culture of RSV-specific B cells isgenerated, which cells remain capable of producing RSV-specificantibodies. These RSV-specific B cells that co-express BCL6 and Blimp-1are preferably further stabilized through the addition of theanti-apoptotic gene Bcl-xL. With the introduction of Bcl-xL it is nowpossible to grow plasmablasts under conditions of low cell density.Hence, the invention also provides a method to culture plasmablastsunder conditions of low cell density comprising generating anRSV-specific antibody producing cell with expression levels of BCL6,Blimp-1 and Bcl-xL with any of the herein described methods.

The amount of BCL6 expression product (preferably a BCL6 protein) in anRSV-specific antibody producing cell is regulated in a variety of ways.

In one embodiment an antibody producing cell is provided with a compoundcapable of directly or indirectly influencing BCL6 expression. Anantibody producing cell is preferably provided with a compound capableof enhancing BCL6 expression, in order to counteract downregulation ofBCL6 during expression of Blimp-1. Such compound preferably comprises aSignal Transducer of Activation and Transcription 5 (STAT5) protein or afunctional part, derivative and/or analogue thereof, and/or a nucleicacid sequence coding therefore. STAT5 is a signal transducer capable ofenhancing BCL6 expression. There are two known forms of STAT5, STAT5aand STAT5b, which are encoded by two different, tandemly linked genes.Administration and/or activation of STAT5 results in enhanced BCL6levels. Hence, downregulation of BCL6 by Blimp-1 is at least in partcompensated by upregulation expression of BCL6 by STAT5 or a functionalpart, derivative and/or analogue thereof. Hence, STAT5 or a functionalpart, derivative and/or analogue thereof is capable of directlyinfluencing BCL6 expression. It is also possible to indirectly influenceBCL6 expression. This is for instance done by regulating the amount of acompound which in turn is capable of directly or indirectly activatingSTAT5 and/or regulating STAT5 expression. Hence, in one embodiment theexpression and/or activity of endogenous and/or exogenous STAT5 isincreased. It is for instance possible to indirectly enhance BCL6expression by culturing an antibody producing cell in the presence ofinterleukin (IL) 2 and/or IL 4 which are capable of activating STAT5.

In one embodiment, an RSV-specific antibody producing cell is providedwith a nucleic acid sequence encoding STAT5 or a functional part,derivative and/or analogue thereof, wherein said nucleic acid sequenceis constitutively active, meaning that STAT5 is continuously expressed,independent of the presence of (endogenous) regulators. In case thatendogenous STAT5 expression is low, or absent, an exogenousconstitutively active nucleic acid sequence encoding STAT5 or afunctional part, derivative and/or analogue thereof is preferablyapplied resulting in a concentration of STAT5 or a functional part,derivative and/or analogue thereof which is sufficient to enhance BCL6expression. Most preferably, an RSV-specific antibody producing cell isprovided with a nucleic acid sequence encoding a compound comprisingSTAT5 or a functional part, derivative and/or analogue thereof,preferably a fusion protein, whose activity is regulated by an exogenousinducer of repressor, so that the extent of activation of BCL6expression is regulated at will. Another system that allows forinduction of BCL-6 is provided by a Tet-on system in which addition oftetracycline and/or derivatives of tetracycline induce activity of atransactivator that induced BCL6 gene transcription followed by BCLprotein synthesis. In one preferred embodiment, an antibody producingcell is provided with a nucleic acid sequence encoding an estrogenreceptor (ER) and STAT5 as a fusion protein ER-STAT5. This fusionprotein is inactive because it forms a complex with heat shock proteinsin the cytosol. This way, STAT5 is unable to reach the nucleus and BCL6expression is not enhanced. Upon administration of the exogenous inducer4 hydroxy-tamoxifen (4HT), the fusion protein ER-STAT5 dissociates fromthe heat shock proteins, so that STAT5 is capable of entering thenucleus and activating BCL6 expression.

Additionally, or alternatively, BCL6 expression in an RSV-specificantibody producing cell is enhanced by culturing said antibody producingcell in the presence of a compound capable of directly or indirectlyenhancing BCL6 expression.

One embodiment therefore provides a method for producing an RSV-specificantibody producing cell comprising:

providing an RSV-specific antibody producing cell with a compoundcapable of directly or indirectly enhancing BCL6 expression; and/or

culturing an RSV-specific antibody producing cell in the presence of acompound capable of directly or indirectly enhancing BCL6 expression.Said compound capable of directly or indirectly enhancing BCL6expression preferably comprises STAT5 or a functional part, derivativeand/or analogue thereof. Provided is therefore a method according to theinvention comprising providing said RSV-specific antibody producing cellwith STAT5 or a functional part, derivative and/or analogue thereof, orwith a nucleic acid sequence encoding STAT5 or a functional part,derivative and/or analogue thereof. In one embodiment said antibodyproducing cell is cultured after introduction of a nucleic acid sequenceencoding STAT5 or a functional part, derivative and/or analogue thereofinto said cell. Said nucleic acid sequence is for instance introducedinto said cell by transfection and/or virus-mediated gene transfer. Manyalternative methods for introducing a nucleic acid sequence into a cellare available in the art which need no further explanation here.

With a compound capable of directly or indirectly enhancing BCL6expression it is possible to enhance expression of endogenous BCL6. Inone preferred embodiment however an antibody producing cell is providedwith a nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof. As explained herein before, anexogenous nucleic acid encoding BCL6 is preferred because this allowsregulation of a BCL6 concentration within a cell independently fromexpression of endogenous BCL6. Hence, even if expression of endogenousBCL6 is low or absent, for instance caused by Blimp-1, an exogenousnucleic acid sequence encoding BCL6 or a functional part, derivativeand/or analogue thereof is still capable of producing a concentration ofBCL6 which is sufficient for influencing the stability of an antibodyproducing cell. Also provided is therefore a method according to theinvention comprising providing an RSV-specific antibody producing cellwith a nucleic acid sequence encoding BCL6 or a functional part,derivative and/or analogue thereof. Preferably, said antibody producingcell is provided with a constitutively active nucleic acid sequenceencoding BCL6 or a functional part, derivative and/or analogue thereof,so that BCL6 expression is maintained even when endogenous BCL6expression of said cell is inhibited by an endogenous repressor such asBlimp-1. Most preferably, expression of said nucleic acid sequenceencoding BCL6 or a functional part, derivative and/or analogue thereofis regulated by an exogenous inducer of repressor, so that the extent ofBCL6 expression is regulated at will. For instance, an induciblepromoter system is used such as a Tet-on or Tet-off system, as alreadydescribed.

In another preferred embodiment, the invention provides a method whereinthe amount of BCL6 is indirectly regulated by providing an RSV-specificantibody producing cell with a nucleic acid sequence encoding E47 or afunctional part, derivative and/or analogue thereof E47 encodes atranscription factor that belongs to a family of helix-loop-helixproteins, named E-proteins. There are four E-proteins, E12, E47, E2-2and HEB, which are involved in lymphocyte development. E12 and E47 areencoded by one gene, named E2A, which is spliced differently. E-proteinscan be inhibited by the E protein inhibitor Id2, and Id3, and by ABF-1(Mathas S., 2006). E proteins have been described as tumor suppressorsand overexpression has been shown to induce apoptosis. One of thespecific targets of E47 are the Socs1 and Socs3 genes. Those Socs genesare known as negative regulators of STAT5b and thus indirectly of BCL6.In other words, expression of E47 within a B cell enhances Blimp-1expression which results in B-cell differentiation towards an antibodyproducing phenotype (plasmacell).

The amount of Blimp-1 expression in an RSV-specific antibody producingcell is also regulated in a variety of ways. In one embodiment anRSV-specific antibody producing cell is provided with a compound capableof directly or indirectly influencing Blimp-1 expression. Additionally,or alternatively, an antibody producing cell is cultured in the presenceof a compound capable of directly or indirectly influencing Blimp-1expression. Further provided is therefore a method according to theinvention comprising providing an RSV-specific antibody producing cellwith a compound capable of directly or indirectly influencing Blimp-1expression. Further provided is a method according to the inventioncomprising culturing said antibody producing cell in the presence of acompound capable of directly or indirectly influencing Blimp-1expression. Preferably, a compound is used that is capable of enhancingBlimp-1 expression in order to counteract downregulation of Blimp-1during expression of BCL6. Said compound most preferably comprisesIL-21.

In one preferred embodiment said compound capable of directly orindirectly influencing Blimp-1 expression comprises a Signal Transducerof Activation and Transcription 3 (STAT3) protein or a functional part,derivative and/or analogue thereof, and/or a nucleic acid sequencecoding therefore. STAT3 is a signal transducer which is involved in Bcell development and differentiation. STAT3 is capable of upregulatingBlimp-1 expression, Further provided is therefore a method according tothe invention wherein said compound capable of directly or indirectlyinfluencing Blimp-1 expression comprises STAT3 or a functional part,derivative and/or analogue thereof, or a nucleic acid sequence encodingSTAT3 or a functional part, derivative and/or analogue thereof. Mostpreferably, expression of said nucleic acid sequence encoding STAT3 or afunctional part, derivative and/or analogue thereof is regulated by anexogenous inducer of repressor, so that the extent of STAT3 expressionis regulated at will. For instance, an inducible promoter system is usedsuch as for instance a Tet-on or Tet-off system. In one embodiment afusion product comprising of STAT3, a derivative or analogue, and ER isintroduced in said cell allowing regulation of STAT3 expression byhydroxytamoxifen.

Since STAT3 is capable of influencing Blimp-1 expression, it is alsopossible to indirectly regulate Blimp-1 expression by administering acompound capable of directly or indirectly regulating the activityand/or expression of STAT3. In one embodiment an antibody producing cellis provided with a compound that is capable of enhancing the activity ofSTAT3, so that Blimp-1 expression is indirectly enhanced as well.Further provided is therefore a method according to the invention,wherein an antibody producing cell is provided with a compound capableof directly or indirectly enhancing activity of STAT3.

Hence, in one embodiment an antibody producing cell is provided with acompound capable of directly or indirectly activating STAT3, in order toenhance Blimp-1 expression.

STAT3 is activated in a variety of ways. Preferably, STAT3 is activatedby providing an antibody producing cell with a cytokine. Cytokines,being naturally involved in B cell differentiation, are very effectivein regulating STAT proteins. Very effective activators of STAT3 areIL-21 and IL-6, but also IL-2, IL-7, IL-10, IL-15 and IL-27 are known toactivate STAT3. Moreover, Toll-like receptors (TLRs) which are involvedin innate immunity are also capable of activating STAT3. One embodimenttherefore provides a method of the invention, wherein said compoundcapable of directly or indirectly influencing Blimp-1 expressioncomprises IL-21, IL-2, IL-6, IL-7, IL-10, IL-15 and/or IL-27. Mostpreferably IL-21 is used, since IL-21 is particularly suitable forinfluencing the stability of an antibody producing cell. IL-21 iscapable of upregulating Blimp-1 expression even when Blimp-1 expressionis counteracted by BCL6.

Additionally, or alternatively a mutated Janus kinase (JAK) is used inorder to activate STAT3. Naturally, a JAK is capable of phosphorylatingSTAT3 after it has itself been activated by at least one cytokine. Amutated Janus kinase capable of activating STAT3, independent of thepresence of cytokines, is particularly suitable in a method according tothe present invention.

As already explained before, a compound capable of enhancing Blimp-1expression in one embodiment comprises a nucleic acid sequence encodingSTAT3 or a functional part, derivative and/or analogue thereof. Thepresence of an exogenous nucleic acid sequence encoding STAT3 or afunctional part, derivative and/or analogue thereof allows for acontinuous presence of STAT3 or a functional part, derivative and/oranalogue thereof even when expression of endogenous STAT3 is very low orabsent.

It is also possible to decrease expression and/or activity of STAT5 inorder to upregulate Blimp-1. If the amount and/or activity of STAT5 isdecreased, activation of BCL6 expression is decreased as well, whichresults in a decreased amount of BCL6 expression product. Since BCL6 andBlimp-1 counteract each other's expression, a decreased amount of BCL6expression product results in an increased amount of Blimp-1 expressionproduct. Compounds capable of downregulating the activity of STAT5 arethus capable of indirectly upregulating Blimp-1. Such compounds forinstance comprise members of the suppressor of cytokine signalling(SOCS) proteins. In one embodiment the amount of Blimp-1 expressionproduct in an RSV-specific antibody producing cell is thereforeupregulated by providing said cell with a SOCS protein, and/or byactivating a SOCS protein within said cell.

In one preferred embodiment the expression and/or activity of STAT5 isdecreased when an RSV-specific antibody-producing cell is provided witha nucleic acid sequence encoding E47 or a functional part, derivativeand/or analogue thereof. Expression of E47 within B cells expressinghigh levels of STAT5b intervenes with differentiation and proliferation,i.e. blocking of STAT5 via E47 and SOCS results in decreased BCL6 levelsand subsequently in increased Blimp-1 levels. Upregulated levels ofBlimp-1 result in a decreased proliferation and in a differentiation ofthe involved cell towards an antibody-producing cell. In other words,expression of E47 within a B cell enhances Blimp-1 expression whichresults in B-cell differentiation towards an antibody producingphenotype (plasma cell).

By at least a functional part of a STAT5 protein, a STAT3 protein,Bcl-xL and/or BCL6 is meant a proteinaceous molecule that has the samecapability—in kind, not necessarily in amount—of influencing thestability of an antibody producing cell as compared to a STAT5 protein,a STAT3 protein, Bcl-xL and/or BCL6, respectively. A functional part ofa STAT5 protein or a STAT3 protein is for instance devoid of amino acidsthat are not, or only very little, involved in said capability. Aderivative of a STAT5 protein, a STAT3 protein, Bcl-xL and/or BCL6 isdefined as a protein which has been altered such that the capability ofsaid protein of influencing the stability of an antibody producing cellis essentially the same in kind, not necessarily in amount. A derivativeis provided in many ways, for instance through conservative amino acidsubstitution wherein one amino acid is substituted by another amino acidwith generally similar properties (size, hydrophobicity, etc), such thatthe overall functioning is likely not to be seriously affected. Aderivative for instance comprises a fusion protein, such as a STAT5-ERor STAT3-ER fusion protein whose activity depends on the presence of 4hydroxy-tamoxifen (4HT). An analogue of a STAT5 protein, a STAT3protein, Bcl-xL and/or BCL6 is defined as a molecule having the samecapability of influencing the stability of an antibody producing cell inkind, not necessarily in amount. Said analogue is not necessarilyderived from said STAT5 protein, STAT3 protein, Bcl-xL and/or BCL6.

In one preferred embodiment said RSV-specific antibody producing cell iscultured in the presence of IL-21 before said antibody producing cell isprovided with a nucleic acid sequence encoding BCL6 or a functionalpart, derivative and/or analogue thereof. Culturing RSV-specificantibody producing cells, preferably B cells, in the presence of IL-21before said cell is provided with a nucleic acid sequence encoding BCL6or a functional part, derivative and/or analogue thereof is preferred,because in these embodiments stability, proliferation and/or antibodyproduction is particularly well improved.

In a preferred embodiment, the invention provides a method forinfluencing the stability of an RSV-specific antibody producing cell asdescribed herein, further comprising directly or indirectly increasingthe amount of Bcl-xL expression product within said antibody producingcell. This is for example accomplished by providing said antibodyproducing cell with a nucleic acid sequence encoding Bcl-xL or afunctional part, derivative and/or analogue thereof or with nucleic acidsequences encoding other anti-apoptotic genes including but not limitedto Bcl-2. In yet another embodiment this is accomplished by providingsaid antibody producing cell with a compound capable of directly orindirectly enhancing Bcl-xL expression, preferably said compoundcomprises APRIL, BAFF, CD40, BCR stimulation, cytokines, growth factorsor downstream effectors like JNK and AKT (PKB).

Bcl-xL is a member of the anti-apoptotic Bcl-2 family, Bcl2-proteinsinteract with and counteract so-called Bcl-2 homology domain 3(BH3)-only family members such as Bax, Bak, Bim, and Bad, which inducecytochome c release following intrinsic death stimuli (Boise, L. H.,1993). Thus, protection of mitochondrial membrane integrity throughproteins like Bcl-xL is critical for cell survival.

STAT5 activation has been shown to protect cells from cell death. STAT5has been shown to regulate the expression of Bcl-xL, supporting ananti-apoptotic role for STAT5. STAT5 positively regulates the Bcl-xLexpression through STAT binding elements within the Bcl-xL promoter. Invivo, Bcl-xL expression is absent in bone marrow of STAT5A/B-doublydeficient mice. Furthermore, STAT5-mediated erythroblast survival isdependent upon upregulation of Bcl-xL. Recently, it has been shown thattransgenic overexpression of Bcl-xL in mouse B cells promotes B cellsurvival and nonmalignant plasma cell foci.

A method according to the invention is particularly suitable forproducing a cell culture comprising RSV-specific antibody producingcells that are capable of proliferating and secreting antibody. In oneembodiment, an RSV-specific memory B cell is used in order to produce anex vivo B cell culture. Said memory B cell is preferably human so thathuman antibodies are produced. Said B cell preferably originates from anindividual, which individual had been previously exposed to RespiratorySyncytial Virus. In one embodiment RSV-specific B cells are isolatedfrom a peripheral blood sample and/or a tonsil sample, using methodsknown in the art. Memory B cells are for instance isolated by selection(magnetic beads sorting) for the B cell marker CD 19 and/or CD22 and(subsequent) selection for cell surface IgG and/or CD27 and/or bynegative selection for IgM, IgD and/or IgA. In a germinal center B cell,BCL6 expression is high whereas Blimp-1 expression is low. Naturaldevelopment into an antibody secreting cell involves upregulation ofBlimp-1 expression. Since Blimp-1 represses BCL6 expression,upregulation of Blimp-1 results in downregulation of BCL6 in a naturalsituation. In a preferred embodiment of the present invention however,Blimp-1 expression is upregulated while BCL6 expression is at least inpart maintained. This results in an RSV-specific antibody producing cellwherein BCL6 and Blimp-1 are co-expressed. Said RSV-specific antibodyproducing cell is capable of proliferating and secreting anti-RSVantibodies and is therefore suitable for use in an ex vivo B cellculture. In a further preferred embodiment, said antibody producing cellis protected by apoptosis by Bcl-xL. An RSV-specific antibody producingcell according to the present invention provides the advantage that itis stable and does not undergo terminal differentiation during aprolonged period. Said antibody producing cell according to theinvention is stable for at least one week, preferably for at least onemonth, more preferably for at least three months, most preferably for atleast six months. A B cell according to the invention is preferablycultured in the presence of CD40L since replication of most B cells isfavoured by CD40L.

In one embodiment BCL6 expression is maintained at essentially the samelevel, or at a higher level, as compared to a germinal center B cellsince a significant BCL6 expression, together with Blimp-1 expression,results in an antibody producing cell with preferred proliferation andantibody production properties and/or stability. In a preferredembodiment, said BCL6 expression and/or Blimp-1 expression areaccompanied by Bcl-xL expression, resulting in even more preferredproliferation and antibody production properties and/or stability.

One embodiment therefore provides a method for producing an RSV-specificantibody producing cell which is stable for at least one week,preferably for at least one month, more preferably for at least threemonths, more preferably for at least six months, the method comprising:

providing an RSV-specific memory B cell;

increasing an expression level of Blimp-1 in said cell; and

increasing and/or maintaining a BCL6 expression level in said cell. Anex vivo method for producing an RSV-specific antibody producing cellcomprising increasing an expression level of Blimp-1 in an RSV-specificmemory B cell and increasing and/or maintaining a BCL6 expression levelin said cell is also provided. Said BCL6 and Blimp-1 expression levelsare preferably brought to, and/or maintained at, essentially the samelevel, or at a higher level, as compared to a plasmablast. In apreferred embodiment said B cell is transduced with BCL6 and Bcl-xL.Further provided is therefore a method for producing an RSV-specificantibody producing cell which is stable for at least three months,comprising:

providing a B cell capable of producing RSV-specific antibodies withBCL6, or a functional part, derivative and/or analogue thereof; and

providing said B cell with Bcl-xL or a functional part, derivativeand/or analogue thereof; and

culturing said B cell.

Said B cell is preferably provided with a nucleic acid sequence encodingBCL6, or a functional part, derivative and/or analogue thereof, and witha nucleic acid sequence Bcl-xL or a functional part, derivative and/oranalogue thereof.

Said B cell is preferably cultured in the presence of a compound capableof enhancing Blimp-1 expression, such as for instance IL-21, IL-2, IL-6,IL-7, IL-10, IL-15, IL-27, or a mutated Janus kinase. Preferably, IL-21is used because this cytokine is particularly suitable for enhancingBlimp-1 expression and stabilizing an antibody producing cell with amethod according to the present invention. Moreover, in order to enhancetransduction efficacy, said B cell is preferably cultured in thepresence of IL-21 before said B cell is transduced with a nucleic acidsequence encoding BCL6 and/or Bcl-xL, or a functional part, derivativeand/or analogue thereof.

In one embodiment said B cell is provided with a SOCS protein or afunctional part, derivative and/or analogue thereof, or a nucleic acidcoding therefore, since a SOCS protein or a functional part, derivativeand/or analogue thereof is capable of indirectly enhancing Blimp-1expression. In another alternative or additional embodiment, said B-cellis provided with E47 or a functional part, derivative and/or analoguethereof, or a nucleic acid coding therefore. As already outlinedearlier, as a result of an increased level of E47 or a functional part,derivative and/or analogue thereof, SOCS protein function is enhancedand Blimp-1 expression is indirectly increased.

In the Examples particularly preferred embodiments are shown. Accordingto one particularly preferred embodiment, RSV-specific B cells arefirstly cultured in the presence of IL-21. Subsequently the B cells aresubjected to a transduction reaction using a nucleic acid encoding BCL6and a nucleic acid encoding Bcl-xL. Preferably spin transduction isused. Most preferably, B cells and virus comprising at least one nucleicacid of interest are mixed, where after the mixture is spinned in orderto achieve a high transduction efficacy. After transduction, the B cellsare cultured in the absence of IL-21 and in the presence of IL-4 andL-cells during 3-5 days in order to allow BCL6 expression. Subsequently,according to this preferred embodiment, the B cells are subjected againto a transduction reaction using a nucleic acid encoding BCL6 and anucleic acid encoding Bcl-xL. Afterwards, the B cells are again culturedin the absence of IL-21 and in the presence of IL-4 and L-cells during3-5 days in order to allow BCL6 expression. Subsequently, cellsexpressing BCL6 and Bcl-xL are isolated and IL-21 is administered againto the culture in order to enhance replication and antibody production.Antibodies that are secreted by Bcl-6, Blimp 1 and Bcl-XL expressingcells in the culture supernatant are preferably screened for in vitroneutralizing capacity/activity/reactivity to RSV. Antibody producingcells that produce those antibodies are preferably further selected, forinstance by limiting dilution culture. Stable RSV-specific B cells arethus obtained wherein BCL6 and Blimp-1 are co-expressed. Said B cellsare capable of replicating and producing antibody in an in vitro cultureduring at least six months,

One embodiment provides a method according to the invention furthercomprising selecting and/or isolating an RSV-specific antibody or afunctional equivalent thereof. In one embodiment IgM producing cells andIgG producing cells are selected and/or isolated. Preferably an IgGproducing cell is selected and/or isolated,

RSV-specific antibody producing cells generated with a method accordingto the invention are suitable for producing antibodies against RSV. Inone preferred embodiment however, the genes encoding the Ig heavy and/orlight chains are isolated from said cell and expressed in a second cell,such as for instance cells of a Chinese hamster ovary (CHO) cell line or293(T) cells. Said second cell, also called herein a producer cell, ispreferably adapted to commercial antibody production. Proliferation ofsaid producer cell results in a producer cell line capable of producingRSV-specific antibodies. Preferably, said producer cell line is suitablefor producing compounds for use in humans. Hence, said producer cellline is preferably free of pathogenic agents such as pathogenicmicroorganisms.

A method according to the invention is preferably used for generating anantibody producing cell that is stable for at least one week, preferablyat least one month, more preferably at least three months, morepreferably at least six months so that commercial antibody productionhas become possible. Most preferably a stable cell line capable ofproducing monoclonal antibodies is produced. This is preferablyperformed by using memory B cells that have for instance been isolatedfrom a sample by selection for CD 19 and/or CD22 (B cell marker) andcell surface IgG and/or CD27 (to mark memory cells) and/or by negativeselection for IgM, IgD and/or IgA. Furthermore, an RSV-specific antibodyproducing cell is for instance selected in a binding assay using RSV ora component derived from RSV, such as for instance the RSV F protein, Gprotein and/or SH protein. Subsequently, according to this preferredembodiment Blimp-1 and BCL6 are co-expressed in said RSV-specificantibody producing cell, resulting in a culture of cells capable ofspecifically binding (a component of) RSV. In yet another preferredembodiment, said B cell is further provided with Bcl-xL or a functionalpart, derivative and/or analogue thereof.

If only one memory cell is used, a cell line according to the inventionwhich produces monoclonal antibodies is obtained. It is also possible togenerate a monoclonal antibody producing cell line starting with B cellscapable of producing antibodies against RSV. After a stable B cellculture has been produced with a method according to the invention, a Bcell capable of producing antibodies against a specific antigen of RSVis isolated and at least a functional part of a gene encoding the Igheavy chain and/or light chain from said B cell is preferably expressedin a second cell line. Preferably at least a functional part of the geneencoding the Ig heavy chain and at least a functional part of the geneencoding the Ig light chain from said B cell are expressed in a secondcell line.

In one embodiment an antibody producing cell, preferably but notnecessarily a memory B cell, that has been obtained from an individualwhich had been previously exposed to RSV, is used in a method accordingto the invention. This way, it has become possible to produce humanantibodies of interest ex vivo.

Further provided is therefore a method for producing antibodies whichare capable of specifically binding and/or neutralizing RespiratorySyncytial Virus, the method comprising:

producing an antibody producing cell capable of producing RSV-specificantibodies with a method according to the invention; and

obtaining antibodies produced by said antibody producing cell.

An isolated or recombinant antibody, as well as an isolated orrecombinant antibody producing cell, obtainable by a method according tothe invention, or a functional equivalent thereof, is also provided.Said antibody preferably comprises antibody D25, AM14, AM16 and/or AM23,or a functional part, derivative or analogue thereof.

Once an RSV-specific antibody producing cell according to the inventionis obtained, at least a functional part of a gene encoding the Ig heavychain and/or light chain of said cell is preferably isolated and/orgenerated artificially. In one embodiment a nucleic acid sequencecomprising at least a functional part of a nucleic acid sequence asdepicted in FIG. 11A, FIG. 12, FIG. 14A, FIG. 14B and/or FIG. 14C isprovided. Said functional part preferably comprises at least one nucleicacid sequence as depicted in FIG. 11A, FIG. 12, FIG. 14A, FIG. 14Band/or FIG. 14C. Said functional part preferably encodes at least oneCDR as depicted in FIGS. 11B and 11C, FIG. 12, FIG. 14A, FIG. 14B and/orFIG. 14C.

Further provided is an isolated, synthetic or recombinant nucleic acidsequence comprising a heavy chain sequence which is at least 70%,preferably at least 80%, more preferably at least 90% homologous to atleast part of the sequence

(SEQ ID NO: 59) CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAA, (SEQ ID NO: 60)ACTATATTATCAAC, (SEQ ID NO: 61)TGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGA, (SEQ ID NO: 62)GGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGG C, (SEQ ID NO: 63)AGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACG, (SEQ ID NO: 64)GAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAA C, (SEQ ID NO: 65)TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG, and/or (SEQ ID NO: 9)CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATGGTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAAACTATATTATCAACTGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGAGGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGACGGAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG,said part having at least 15 nucleotides. Said heavy chain sequence ispreferably derived from antibody D25. Said heavy chain sequencepreferably comprises a sequence which is at least 70%, preferably atleast 80%, more preferably at least 90% homologous to a sequence asdepicted in FIG. 11A. An isolated, synthetic or recombinant nucleic acidsequence comprising a heavy chain sequence consisting of any of theabove mentioned heavy chain sequences is also herewith provided.

An isolated, synthetic or recombinant nucleic acid sequence comprising alight chain sequence which is at least 70%, preferably at least 80%,more preferably at least 90% homologous to a least part of the sequence

(SEQ ID NO: 66) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCATCACTTGC, (SEQ ID NO: 67) CAGGCGAGTCAGGACATTGTCAACTATTTAAAT,(SEQ ID NO: 68) TGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAC,(SEQ ID NO: 69) GTTGCATCCAATTTGGAGACA, (SEQ ID NO: 70)GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGT, (SEQ ID NO: 71)CAACAATATGATAATCTCCCA, (SEQ ID NO: 72)CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA and/or (SEQ ID NO: 10)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTGTCAACTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGTTGCATCCAATTTGGAGACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGTCAACAATATGATAATCTCCCACTCACATTCGGCGGAGGGACC AAGGTTGAGATCAAAAGA,said part having at least 15 nucleotides, is also provided. Said lightchain sequence is preferably derived from antibody D25.

Said light chain sequence preferably comprises a sequence which is atleast 70%, preferably at least 80%, more preferably at least 90%homologous to a sequence as depicted in FIG. 11A. An isolated, syntheticor recombinant nucleic acid sequence comprising a heavy chain sequenceconsisting of any of the above mentioned light chain sequences is alsoherewith provided.

Further provided is an isolated, synthetic or recombinant nucleic acidsequence comprising a heavy chain sequence which is at least 70%,preferably at least 80%, more preferably at least 90% homologous to atleast part of the sequence

(SEQ ID NO: 94) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCGGCCTCT, (SEQ ID NO: 95) GGATTCAGCTTCAGTCACTATGCC,(SEQ ID NO: 96) ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGT T,(SEQ ID NO: 97) ATATCTTATGATGGAGAAAATACA, (SEQ ID NO: 98)TATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCT CTATATTACTGT,(SEQ ID NO: 99) GCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC,(SEQ ID NO: 100) TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCAG and/or(SEQ ID NO: 101) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCAGCTTCAGTCACTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCTTATGATGGAGAAAATACATATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAACACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACTGTGCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGGCCACGGTC ACCGTCTCCTCA,said part having at least 15 nucleotides. Said heavy chain sequence ispreferably derived from antibody AM14. An isolated, synthetic orrecombinant nucleic acid sequence comprising a heavy chain sequenceconsisting of any of the above mentioned heavy chain sequences is alsoherewith provided.

An isolated, synthetic or recombinant nucleic acid sequence comprising alight chain sequence which is at least 70%, preferably at least 80%,more preferably at least 90% homologous to a least part of the sequence

(SEQ ID NO: 102) GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGT, (SEQ ID NO: 103) CAGGACATTAAGAAGTAT,(SEQ ID NO: 104) TTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCA C,(SEQ ID NO: 105) GATGCATCC, AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTGAAGATA TTGGAACATATTACTGT,(SEQ ID NO: 106) CAACAGTATGATAATCTGCCTCCGCTCACT, (SEQ ID NO: 107)TTCGGCGGAGGGACCAAGGTGGAGATCAAAC and/or (SEQ ID NO: 108)GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAAGAAGTATTTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGTCAACAGTATGATAATCTGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG,said part having at least 15 nucleotides, is also provided. Said lightchain sequence is preferably derived from antibody AM14. An isolated,synthetic or recombinant nucleic acid sequence comprising a heavy chainsequence consisting of any of the above mentioned light chain sequencesis also herewith provided.

Further provided is an isolated, synthetic or recombinant nucleic acidsequence comprising a heavy chain sequence which is at least 70%,preferably at least 80%, more preferably at least 90% homologous to atleast part of the sequence

(SEQ ID NO: 109) GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT, (SEQ ID NO: 110) GGATTCACATTCAGTAGTTATAAC,(SEQ ID NO: 111) ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACA C,(SEQ ID NO: 112) ATTAGTGCGGGTAGTAGTTACATA, (SEQ ID NO:113)TACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCT GTGTATTACTGT,(SEQ ID NO: 114) GCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCC, (SEQ ID NO: 115) TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and/or(SEQ ID NO: 116) GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACATTCAGTAGTTATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACACATTAGTGCGGGTAGTAGTTACATATACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAACTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACTGTGCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCCTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCA,said part having at least 15 nucleotides. Said heavy chain sequence ispreferably derived from antibody AM16. An isolated, synthetic orrecombinant nucleic acid sequence comprising a heavy chain sequenceconsisting of any of the above mentioned heavy chain sequences is alsoherewith provided.

An isolated, synthetic or recombinant nucleic acid sequence comprising alight chain sequence which is at least 70%, preferably at least 80%,more preferably at least 90% homologous to a least part of the sequence

(SEQ ID NO: 117) CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTCCTGCACTGGGAGC, (SEQ ID NO: 118) AGCTCCAACATCGGGGCAGGTTATGAT,(SEQ ID NO: 119) GTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTA T,(SEQ ID NO: 120) GGCAACACT, AATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATG TAGGCTGATTATACTGC,(SEQ ID NO: 121) CACTCCTATGACAGAAGCCTGAGTGGT, (SEQ ID NO: 122)TCAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAG and/or (SEQ ID NO: 123)CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGCAACACTAATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGCCACTCCTATGACAGAAGCCTGAGTGGTTCAGTATTCGGCGGAGGGACCAAGCTGACCGTC,said part having at least 15 nucleotides, is also provided. Said lightchain sequence is preferably derived from antibody AM16. An isolated,synthetic or recombinant nucleic acid sequence comprising a heavy chainsequence consisting of any of the above mentioned light chain sequencesis also herewith provided.

Further provided is an isolated, synthetic or recombinant nucleic acidsequence comprising a heavy chain sequence which is at least 70%,preferably at least 80%, more preferably at least 90% homologous to atleast part of the sequence

(SEQ ID NO: 124) CAGGTGCAACTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTCCTGTGCAGCGACT, (SEQ ID NO: 125) GGATTCAACTTCCATAACTACGGC,(SEQ ID NO: 126) ATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGT T,(SEQ ID NO: 127) GTTTGGTATGATGGAAGTAAGAAA, (SEQ ID NO: 128)TACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCT GTTTATTATTGT,(SEQ ID NO: 129) GTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCC,(SEQ ID NO: 130) TGGGGCCAGGGAACCCTGGTCACCGTATCCTCAG and/or(SEQ ID NO: 131) GAGGTGCAGCTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGACTGTCCTGTGCAGCGACTGGATTCAACTTCCATAACTACGGCATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTTGTTTGGTATGATGGAAGTAAGAAATACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATTGTGTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCG AGT,said part having at least 15 nucleotides. Said heavy chain sequence ispreferably derived from antibody AM23. An isolated, synthetic orrecombinant nucleic acid sequence comprising a heavy chain sequenceconsisting of any of the above mentioned heavy chain sequences is alsoherewith provided.

An isolated, synthetic or recombinant nucleic acid sequence comprising alight chain sequence which is at least 70%, preferably at least 80%,more preferably at least 90% homologous to a least part of the sequence

(SEQ ID NO: 132) TCCTATGTGCTGACTCAGCCACCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCACCTGTGGAAGAAAC, (SEQ ID NO: 133) AACATTGGAAGTGAAACT,(SEQ ID NO: 134) GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTA T,(SEQ ID NO: 135) GATGATGAC, GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATG AGGCCGACTATTACTGT,(SEQ ID NO: 136) CAGGTGTGGGATAGGAGTAATTATCATCAGGTA, (SEQ ID NO: 137)TTCGGCGGAGGGACCAAGTTGACCGTCCTAG and/or (SEQ ID NO: 138)TCCTATGTGCTGACTCAGCCCCCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGCGATCACCTGTGGAAGAAACAACATTGGAAGTGAAACTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATGACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGGAGTAATTATCATCAGGTATTCGGCGGAGGG ACCAAGCTGACCGTC,said part having at least 15 nucleotides, is also provided. Said lightchain sequence is preferably derived from antibody AM23. An isolated,synthetic or recombinant nucleic acid sequence comprising a heavy chainsequence consisting of any of the above mentioned heavy chain sequencesis also herewith provided.

A nucleic acid sequence encoding an amino acid sequence which is atleast 70%, preferably at least 80%, more preferably at least 90%identical to at least a functional part of an amino acid sequence asdepicted in FIG. 11, FIG. 14A, FIG. 14B and/or FIG. 14C, said parthaving at least 5 amino acid residues is also provided. Said nucleicacid sequence preferably encodes an amino acid sequence which is atleast 80% identical to heavy chain CDR sequence 1, 2 and/or 3 and/orlight chain CDR sequence 1 or 2 depicted in FIGS. 11B and 11C. Inanother preferred embodiment said nucleic acid sequence encodes an aminoacid sequence which is at least 80% identical to at least one of the CDRsequences depicted in FIG. 14A, in FIG. 14B and/or in FIG. 14C. In onepreferred embodiment said nucleic acid sequence encodes an amino acidsequence which is at least 70% identical to a heavy chain sequencedepicted in FIG. 11A, to a heavy chain sequence depicted in FIG. 14A, toa heavy chain sequence depicted in FIG. 11B, to a heavy chain sequencedepicted in FIG. 14C, to a light chain sequence depicted in FIG. 11A, toa light chain sequence depicted in FIG. 14A, to a light chain sequencedepicted in FIG. 14B, and/or to a light chain sequence depicted in FIG.14C.

Further provided is therefore an isolated, synthetic or recombinantnucleic acid sequence comprising a sequence encoding an amino acidsequence which is at least 70%, preferably at least 80%, more preferablyat least 85% identical to an amino acid sequence as depicted in FIG. 11.Said nucleic acid sequence preferably encodes an amino acid sequencewhich is at least 80% identical to heavy chain CDR sequence 1, 2 and/or3 and/or light chain CDR sequence 1 or 2 as depicted in FIGS. 11B and11C. One embodiment provides an isolated, synthetic or recombinantnucleic acid sequence comprising a sequence encoding an amino acidsequence which is at least 70% identical to the amino acid sequenceNYIIN (SEQ ID NO: 1), and/or at least 75% identical to the sequenceGIIPVLGTVHYAPKFQG (SEQ ID NO: 2), and/or at least 70% identical to thesequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3), and/or at least 85% identicalto the sequence QASQDIVNYLN (SEQ ID NO: 4), and/or at least 70%identical to the sequence VASNLET (SEQ ID NO: 5), and/or at least 70%identical to the sequence

(SEQ ID NO: 7) QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSS,and/or at least 70% identical to the sequence

(SEQ ID NO: 8) DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGT KVEIKRTV.

A nucleic acid sequence according to the invention is preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,most preferably at least 95% homologous to any of the above recitedsequences.

Further provided is an isolated, synthetic or recombinant nucleic acidsequence comprising a sequence encoding an amino acid sequence which isat least 70%, preferably at least 80%, more preferably at least 85%identical to an amino acid sequence as depicted in FIG. 14A-C. Saidnucleic acid sequence preferably encodes an amino acid sequence which isat least 70% identical to a CDR sequence as depicted in FIG. 14A, 14Band/or 14C. One embodiment provides an isolated, synthetic orrecombinant nucleic acid sequence comprising a sequence encoding anamino acid sequence which is at least 70% identical to an amino acidsequence selected from the group consisting of:

(SEQ ID NO: 73) GFSFSHYA, (SEQ ID NO: 74) ISYDGENT, (SEQ ID NO: 75)ARDRIVDDYYYYGMDV, (SEQ ID NO: 76) QDIKKY,  (SEQ ID NO: 77)DAS, QQYDNLPPLT, (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS, (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,(SEQ ID NO: 80) GFTFSSYN, (SEQ ID NO: 81) ISAGSSYI, (SEQ ID NO: 82)AREDYGPGNYYSPNWFDP, (SEQ ID NO: 83) SSNIGAGYD, (SEQ ID NO: 84)GNT, HSYDRSLSG, (SEQ ID NO: 85)EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSSYIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWFDPWGQGTLVTVSS, (SEQ ID NO: 86)QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVSD RFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSV FGGGTKLTV,(SEQ ID NO: 87) GFNFHNYG, (SEQ ID NO: 88) VWYDGSKK, (SEQ ID NO: 89)VRDKVGPTPYFDS, (SEQ ID NO: 90) NIGSET, (SEQ ID NO: 91) DDD, QVWDRSNYHQV,(SEQ ID NO: 92) EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGSKKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVG PTPYFDSWGQGTLVTVSS,and (SEQ ID NO: 93) SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGG TKLTV.

A nucleic acid sequence according to the invention is preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,most preferably at least 95% homologous to any of the above recitedsequences.

As already explained herein before, nucleic acid sequences according tothe present invention are particularly suitable for expressing anantibody or a functional part, derivative or analogue thereof accordingto the invention, preferably D25, AM14, AM16, AM23 or a functional part,derivative and/or analogue thereof, in a nucleic acid expression system.A nucleic acid sequence according to the present invention is preferablyexpressed in a cell, more preferably in a producer cell adapted forantibody production.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

EXAMPLES

Materials and Methods

Maintenance and Isolation of Human B Cells

Using standard procedures, CD19 positive human B cells were isolatedfrom bloodbank derived buffy coat (other sources can be fresh blood withan anticoagulation factor, or a lymphoid organ for example tonsil orspleen). In brief, total peripheral blood mononuclear cells (PBMC) wereisolated using ficoll density separation (Amersham, Buckinghamshire,UK). CD22 labeled beads were used to positively selected B cells by MACScell sorting technique as described by the manufacturer (Miltenyi,Utrecht, Netherlands). Cells were subsequently stained with appropriatecombinations of monoclonal antibodies (mAbs) to CD19, CD27, IgD, IgM andIgA (Becton Dickinson (BD), Franklin Lakes, N.J., USA). Memory B cellsthat are positive for CD 19 and CD27 and negative for IgM, IgA and IgDwere then sorted using the FACSAria (BD) (FIG. 1). Besides memory Bcells, other B cells subsets, like naïve, naïve, follicular, memory,antibody producing, centroblast, centrocyte, germinal center, plasmablast, plasma cell, marginal zone, perisinusoidal or transitional Bcells (many of those subsets have only been determined in mice) can beisolated using appropriate markers.

Cell Culture

Sorted cells were washed and cultured in 24 well plates (1.5 to 2×10⁵cells/ml) on 80 Gray, irradiated CD40L-expressing L-cells (5×10⁴cells/ml; provided by DR. J. Banchereau, Schering Plough France,Dardilly France), in complete medium (Iscove's Modified D MinimalEssential Medium containing 8% fetal calf serum (FCS) andPenicillin/Streptomycin). Unless mentioned otherwise, theseCD40L-expressing L-cells are always present in the cultures incombination with 8% FCS. To prepare the B cell for retroviraltransduction cells were cultured for 36 hours in the presence of mouseIL-21 (50 ng/ml, R&D, Minneapolis, Minn., USA). After transduction cellsare preferentially cultured in the presence of IL-21, however cells dorespond to IL-4, IL-15 and IL-10 (not excluding other cytokines). Forexample, IL-4 induced B cell expansion is lower compared to IL-21 andlower levels of cell division can be required in some experiments.

Retroviral Constructs and Production of Recombinant Retrovirus

Constitutive active mutants of STAT5a and b have been describedpreviously. DNAs encoding these mutants and wildtype STAT5b wereobtained from T. Kitamura (IMSUT, Tokyo, Japan). Bcl-6 was identified ina senescence rescue screen in murine fibroblasts as an inhibitor ofanti-proliferative p19ARF-p53 signaling. Bcl-XL was identified as ananti-apoptose factor, which was kindly provided by Dr Korsmeyer (HowardHughes Medical Institute, Boston, US). These DNAs were ligated intoLZRS-linker-IRES-GFP (or IRES-YFP or IRES-NGFR) vector that wasdescribed previously (Heemskerk et al., 1997; Heemskerk et al., 1999).Instead of the IRES-GFP (Green Fluorescent Protein) marker also anIRES-YFP (Yellow Fluorescent Protein) or an IRES-NGFR (Nerve GrowthFactor Receptor) was used. NGFR is a signaling-incompetent mutant of theNGFR, kindly provided by Dr. C. Bonini A monoclonal antibody againstNGFR (Chromaprobe, Mountain View, Calif., US or Miltenyi) was used tovisualize NGFR-expressing cells.

For production of recombinant retrovirus, the retroviral plasmids weretransfected into a helper-virus free amphotropic producer cell linePhoenix-A, a derivative of the human embryonic kidney cell line 293(Kinsella and Nolan, 1996) (a kind gift of Dr. G. Nolan, StanfordUniversity, Palo Alto, Calif.), using Fugene-6 (Roche DiagnosticsNetherlands, Almere, Netherlands) according to manufacturers protocols.Two days later selection of transfected cells started by the addition of2 μg/ml puromycin (Becton Dickinson Clontech Laboratories, Palo Alto,Calif.). Ten to 14 days after transfection 6×10⁶ cells were plated per10 cm petridish (Becton Dickinson Discovery Labware, Bedford, Mass.) in10 ml complete medium without puromycin. The next day the medium wasrefreshed and on the following day retroviral supernatant was harvested,centrifuged and frozen in cell free aliquots at −70° C. This approachaffords a reproducible rapid, large scale and high titer retroviralproduction of over 3×10⁶ infectious virus particles/ml.

Retroviral Transduction

The recombinant human fibronectin fragments CH-296 transductionprocedure (RetroNectin™; Takara, Otsu, Japan) was performed as describedpreviously (Heemskerk et al., 1997; Heemskerk et al., 1999). Non-tissueculture-treated 24 wells plates (Costar, Badhoevedorp, Netherlands) werecoated with 0.3 ml of 30 μg/ml recombinant human fibronectin fragmentCH-296 at room temperature for 2 hours or overnight at 4° C. Whendifferent sized non-tissue culture plates were used, reagents were usedproportionately. The CH-296 solution was removed, followed by incubationwith 2% human serum albumin (HSA) in phosphate buffered saline (PBS) for30 min at room temperature, followed by washing once with PBS. 5×10⁵ Bcells, which were prepared for retroviral transduction were plated in0.25 ml RPMI without FCS and L-cells and mixed with 0.25 ml of thawedretroviral supernatant. For the Bcl-6 Bcl-XL double transduction 125 μlof Bcl-6-IRES-NGFR (or IRES-YFP) (Shvarts A. et al. Genes Dev., 2002)and 125 μl of Bcl-XL-IRES-GFP (provided by S. Korsmeyer, Howard HughesMedical Institute, Childrens Hospital, Boston, USA) were mixed and addedto the cells. The culture was subsequently centrifuged at 1800 rpm at25° C. for 60 minutes and incubated for 6 hours at 37° C. Next 0.25 mlof supernatant was removed and 0.25 ml of fresh retroviral supernatantwas added. The culture was again centrifuged at 1800 rpm at 25° C. for60 minutes and incubated at 37° C. overnight. The next morning cellswere transferred to 24 wells tissue culture treated plate (Costar) andcultured for 3-5 days under normal conditions in the presence of humanIL-4 (50 ng/ml) or mouse IL-21 (50 ng/ml, R&D, Minneapolis, Minn., USA).Transduction efficiency was determined by antibody staining of atruncated, signaling incompetent mutant of Nerve Growth Factor Receptor(ΔNGFR, provided by C. Bonini, St. Raphael Hospital, Milan, Italy) or(co) expression of GFP and or YFP. The cells containing the transgene(s)of interest are then selected for further experiments.

Flowcytometry

Antibodies against the human molecules IgD, IgG, CD3, CD 19, CD20, CD27,CD38, CD40, CD45, CD56, CD70, CD80, CD86, HLA-DR (BD) directly labeledwith FITC, PE, PERCP, PE-Cy5, APC or APC-Cy7 and IgM, kappa light chain,lambda light chain, CD 138, directly labeled with PE (DAKO) were usedfor flowcytometry analysis. Stained cells were analyzed using a LSRII(BD) and FACS data was processed with Flow Jo computer software (TreeStar, Inc).

Proliferation Experiment

Naïve and memory B cells were isolated from fresh PBMC on the FACSAria:Naïve B cells: CD19-Pe-Cy7 pos, CD27-APC neg, IgD-PE pos

Memory B cells: CD19-Pe-Cy7 pos, CD27-APC pos, IgD-PE neg, IgA-FITC negCells were washed in PBS and resuspended in 0.5 ml RPMI (37° C.) withoutFCS. An equal amount of IMDM containing 2 μM Carboxyfluoresceinsuccinimidyl ester (CFSE) was added to the cell mixture and incubatedfor 7 min at 37° C. Up labeling of the cells was stopped by washing thecell with cold FCS. Cells were resuspended in 500 μl IMDM-8% FCS andcultured with L-cells and in the absence or presence of IL-21.Non-labeled cells were used as control. After 36 hrs (immediately beforetransduction) a proportion of cells was analyzed for their CFSE content.Remaining cells were spin transduced with Bcl-6-IRES-NGFR, cultured for3 days, and analyzed for their CFSE content using the LSRII. Data wasanalyzed using FlowJo software (Treestar)

Isolation of antigen specific human B cells using high speed single cellsorting In addition to the memory B cell isolation method describedabove starting with MBC (i.e. 100 cell/well cultures), human memory Bcells can also be incubated with a fluorescent labeled antigen andsorted based on antigen recognition. An example is the isolation of Bcells that bind phycoerythrin (PE) labeled Tetanus Toxoid (provided byA. Radbruch, Berlin, Germany) (FIG. 4). Cells were cultured at 1cell/well and checked for TT binding. Notwithstanding that any otherlabeled antigen can be used.

Determining the B Cell Receptor (BCR) Expression Alter Long Term Cultureof Bcl-6 and Bcl-XL Transduced Cells

It is known that B cells that differentiate during in vitro culture losetheir BCR membrane expression, which is also observed in EBV transformedB cells. Therefore B cells transduced with Bcl-6 and Bcl-XL and culturedin the presence of IL-21 were stained for GFP, NGFR, CD 19, Kappa and/orLambda or IgG or with labeled Tetanus Toxoid. To show the usefulness ofthe BCR expression we sorted TT-PE (Radbruch) binding cells using theFACSAria (BD) at 1 cell/well in 96-well plates, which were seeded withL-cells and IL-21 containing culture medium. After three weeks TetanusToxoid binding of outgrowing clones was checked using the FACS Canto(BD). Therefore cells were harvested and stained in 96-well plates withGFP, NGFR, CD19 and TT-PE.

Development of Bcl-6 and Bcl-XL Double Positive B Cell Lines thatSecrete Antibodies

B cell lines were created that produces monoclonal antibodies and are100% Bcl-6 and Bcl-XL double positive. First this was achieved byinducing proliferation and differentiation using IL-21. Meanwhile thesecells are transduced with the Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFPretroviruses. The cells are maintained on IL-4 for 3-4 days. The cellsthat are transduced with either one or both retroviruses then expressthe transgene and will therefore express the NGFR or GFP protein. Theexpression of NGFR and/or GFP can be visualized by using the LSRII (BD).If necessary, cells can be transduced again to obtain higher numbers ofcells expressing both transgenes. Irrespective of a second transductionthe cells that express both transgenes are sorted using the FACS Aria(BD) and cultured at a cell density ranging from 10-500 cells/well in96-well plates in the presence of IL-21 and 2500 to 5000 L-cells/well.These mini-bulk-cultures (MBC) secrete relatively large amounts ofantibody in the culture supernatant already at day 5 which then can beused for screening purposes. Screening can be based on techniquesavailable for the antigen of interest e.g. ELISA/EIA/RIA, Western blotor direct functional assays like neutralization of cytokine blockingexperiments. After screening and selection of MBC that recognize theantigen of interest (TT and RSV in our experiments), cells are subclonedat 0.5-1 cell/well in 96 well in the presence of IL-21. Subcloningnormally takes 2-3 weeks and can be performed by limiting dilution (LD)cultures or single cell sorting using flow cytometry (FACSAria).

RSV A-2 Virus Stock and HEp2 cNaïveell Line

The RSV A-2 virus (kindly provided by G. van Bleek, WKZ, Utrecht) andHEp2 cell line (Clinical Laboratory, AMC, Amsterdam), were cultured inlarge quantities and frozen in liquid nitrogen.

The adherent HEp2 cell line was cultured in normal medium in T175 Falconbottles before aliquots were frozen.

To obtain a high titer. RSV stock, HEp2 cells were seeded and culturedto reach 50-60% confluence. The original RSV stock was added (1/20dilution total volume 5 ml) for 45′ at RT on the HEp2 cells. 15 ml freshmedium was added and cells were left o/n at 37° C., 5% CO₂ with thecoverlid open. The next morning culture supernatant was carefullyremoved and 15 ml medium containing 1% FCS was added. Cells were leftfor 24 to 36 hours at 37° C., 5% CO₂ with the coverlid closed. When RSVinduced syncytia were clearly visible and the majority of the syncytiawere still intact, the medium was harvested, filtered (0.22 μm) and spinat 1450 rpm at RT before samples were snap frozen and stored in liquidnitrogen. A second harvest can be obtained by immediately adding newmedium containing 1% FCS and freezing this batch 4-6 hours later.

RSV Lysate for ELISA

HEp2 cells that were infected with RSV A-2 to obtain virus stocks wereused to isolate RSV proteins. First cell were carefully washed with PBSand trypsinized Trypsin (Gibco) was washed away and the cell pellet waslysed with 1% octylglucoside (cell pellet of one Tl 75 flask was treatedwith 2 ml octylglucoside). Suspension was homogenized with syringe andneedle (10 times up and down), incubated for 1 hour on ice and thendialyzed against 2 L TBS buffer pH 7.4, o/n at 4° C. Supernatant wasobtained after spin down of cell debris. The protein content wasdetermined at 3.6 mg/ml and was used at 20 μg/ml (50 μl) in ELISAs.

Determining TCID50 and PFU of RSV Stocks

To determine the TCID50, 10⁴ HEp2 were seeded in 96 well plates andinfected with a 2 or 10 step serial dilution of RSV virus in 4-plo. 2-3days later culture supernatant were removed and cells were fixed with80% acetone for 10′ at RT. After removal of the acetone, the fixed celllayer was dried and kept at 4° C. or frozen at −20° C. To stain RSV HEp2cells the plates were first blocked with 5% milkpower in PBS 0.1% Tween20. Then plates were washed 3 times before being incubated for 3-5 hoursat 37° C. with polyclonal goat anti-RSV-HRP (1:500, Biodesign, Saco,Me., US) and washed extensively. Next the wells were incubated with AECsubstrate for 30′ at RT. Infected foci stain red and can be observed byeye using a light microscope and can be counted. Standard Excel softwarewas used to determine the TCID₅₀.

To determine the amount of plaque forming units (PFU) of the virus,1×10⁵/ml of HEp2 cells in 24 well plates were incubated with 10-foldserial dilutions (10⁻³-10⁻⁷) of RSV virus stock in medium with 1% FCS at37° C. for 45′ (200 μl) before cells and virus were covered with 0.5 mlhand warm 0.25% seaplaque agar (Biozyme). The agarose layer prevents thespreading of the virus to uninfected cells through the culture medium.Thereby the virus can infect only neighboring cells, which eventuallyare killed by the virus creating plaques in the monolayer of HEp2 cells.Those plaques can best be visualized by staining the fixed cells (96%ethanol-100% acetic acid-10% formalin 6:2:1) with 1% crystal violetsolution. Plaques are counted (by at least two different individuals)and the PFU value can be determined.

Selection of Respiratory Syncytial Virus (RSV) Neutralizing Antibodies

To obtain anti-respiratory syncytial virus (RSV) B cell clones,peripheral blood cells (PBMC) from two donors were isolated frombloodbank derived buffy coats (donor B62 and B63). Before sortingCD19^(pos)IgM^(neg)IgD^(neg)IgA^(neg)CD27^(pos) cells using the FACSAria(BD) (FIG. 1), CD22+ cells were isolated using MACS beads and columns(Miltenyi). Only if mentioned differently, cells were cultured withL-cells. Cells were cultured for 36 hours in the presence IL-21 beforebeing transduced with Bcl-6-IRES-NGFR only. After 12 h cells wereharvested and cultured for 3 days in the presence of IL-4 before NGFRexpressing cells were sorted using MACS beads (Miltenyi) and immediatelytransduced with Bcl-XL-IRES-GFP. The B cells that did not bind to theMACS beads were washed and transduced with Bcl-6 and Bcl-XL at the sametime. After 12 h cells were harvested, pooled and cultured for 3 days inthe presence of IL-4 before being sorted on GFP and NGFR expression onthe FACSAria. Cells were washed and cultured at 100 cell/well density in96 well plates (Costar) in the presence of IL-21.

The double transduced Bcl-6 and Bcl-XL B cell cultures were screened forRSV binding using a RSV-infected HEp2 cell lysate ELISA and were testedin parallel using a RSV microneutralization experiment. In brief, 10⁴HEp2 cells arc seeded in flat bottom 96 well plates (Costar) in completemedium. The next day medium is replaced for 1 h at RT with the mixtureof RSV virus and cell culture supernatant which have been pre-incubatedfor 30 min at 37° C. The total volume is 25 μl and the RSV endconcentration is 0.1 MOI. After 1 h the virus supernatant mixture is 9times diluted with PBS and replaced with 100 μl IMDM/5% FCS. After 2days cells are fixed with 80% acetone and stained with polyclonalanti-RSV-HRP (Biodesign). Using H₂O₂ and AEC cells infected with RSVdevelop a red stain. Using light microscopy infected cells can beobserved and counted if necessary. As a control for RSV neutralization agoat polyclonal anti-RSV (Abeam, Cambridge, Mass.) is used.

RT-PCR and Cloning of VH and VL Regions

Total RNA was isolated from ˜5×10 B cells with the RNeasy® mini kit(Qiagen, Venlo, The Netherlands). 250 ng of total RNA was reversetranscribed in a volume of 20 μl containing 1× first strand buffer, 500μM dNTP, 250 ng random hexamers, 5 mM DTT, 40 U RNasin (Promega) and 200U SuperScript III RT (Invitrogen). The cDNA was diluted 10× in Ultrapurewater and 2.5 μl of cDNA was subjected to PCR in a 50 μl solutioncontaining 20 mM Tris-HCL, 50 mM KCL, 2.5 mM MgCl2, 250 μM dNTP, 1 UAmpliTaq Gold DNA polymerase (Applied Biosystems Inc.), and 25 pmol ofeach primer. PCR conditions were as follows: 8 min denaturing step at96° C. followed by 35 cycles of 30 sec at 96° C., 30 sec at 60° C., 1min at 72° C., and a final 10 min extension at 72° C.

PCR products were run on agarose gels, purified and cloned into thepCR2.1 TA cloning vector according to manufacturers' recommendations.Sequence analysis was performed using BigDye Terminator chemistry(Applied Biosystems Inc.) and Vector-NTI software (Invitrogen).

To rule out reverse transcriptase and/or DNA polymerase inducedmutations, several independent cDNA conversions and PCR reactions wereperformed and individually cloned and sequence analyzed. Consensussequences were determined with Vector-NTI Contig Express software.

For recombinant protein antibody expression in 293T cells full lengthheavy and light chain constructs were generated in pcDNA3.1(+)Zeo(Invitrogen). The heavy chain expression vector was constructed by PCRamplification of the heavy chain leader sequence and VH region of cloneD25 introducing a 5′-NheI site and a 3′-XhoI site. The IgG1 constantregion (CH1-hinge-CH2-CH3) was amplified from the same cDNA whileintroducing a 5′-XhoI and a 3′-NotI site. The full length heavy chainexpression vector was obtained by three point ligation into NheI/NotIdigested pcDNA3.1(+)Zeo. The full length light chain expressionconstruct was generated by PCR amplification of the light chain leadersequence, VL region and light chain constant region with primersintroducing a 5′-NheI and 3′-NotI site. The latter product was clonedinto NheI/NotI digested pcDNA3.1(+)Zeo to obtain a full length lightchain expression vector.

Sequence analysis was performed to confirm correctness of the expressionconstructs.

Transient double transfection (Fugene-6, Roche, Germany or LipofectamineLTX, Invitrogen) of 293T cells with both heavy and light chainexpression vectors was performed to produce recombinant monoclonalantibody. A FACS staining with the resulting culture supernatant (48hours) on RSV infected Hep2 cells was performed to show functionalbinding of the antibody to the RSV F-protein.

The oligonucleotides used for PCR amplifications were:

VH regions: VH1-For (SEQ ID NO: 11) 5′-AAATCGATACCACCATGGACTGGACCTGGAGG-3′ VH1B-For (SEQ ID NO: 12) 5′-AAATCGATACCACCATGGACTGGACCTGGAGM-3′ VH2A-For (SEQ ID NO: 13) 5′-AAATCGATACCACCATGGACACACTTTGCTMCAC-3′ VH2B-For (SEQ ID NO: 14) 5′-AAATCGATACCACCATGGACATACTTTGTTCCAAC- 3′ VH3-For (SEQ ID NO: 15) 5′-AAATCGATACCACCATGGAGTTTGGGCTGAGC-3′ VH3B-For (SEQ ID NO: 16) 5′-AAATCGATACCACCATGGARYTKKGRCTBHGC-3′ VH4-For (SEQ ID NO: 17) 5′-AAATCGATACCACCATGAAACACCTGTGGTTCTT-3′ VH5-For (SEQ ID NO: 18) 5′-AAATCGATACCACCATGGGGTCAACCGCCATC-3′ VH6- For (SEQ ID NO: 19) 5′-AAATCGATACCACCATGTCTGTCTCCTTCCTC-3′ Cgamma-Rev (SEQ ID NO: 20) 5′-GGGTCTAGACAGGCAGCCCAGGGCCGCTGTGC-3′ Vkappa regions: Vk1-For(SEQ ID NO: 21) 5′ -AAATCGATACCACCATGGACATGAGGGTCCCY-3′ Vk1B-For(SEQ ID NO: 22) 5′-AAATCGATACCACCATGGACATGAGRGTCCYY-3′ Vk2-For(SEQ ID NO: 23) 5′ -AAATCGATACCACCATGAGGCTCCCTGCTCAG-3′ Vk3-For(SEQ ID NO: 24) 5′ -AAATCGATACCACCATGGAARCCCCAGCGCA-3′ Vk4-For(SEQ ID NO: 25) 5′ -AAATCGATACCACCATGGTGTTGCAGACCCAG-3′ Ck-Rev (SEQ ID NO: 26) 5′ -GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3′Vlambda regions: V11aecb (SEQ ID NO: 27) 5′-AAATCGATACCACCATGGCCTGGTCCCCTCTCCTCC-3′ V11g (SEQ ID NO: 28) 5′-AAATCGATACCACCATGGCCGGCTTCCCTCTCCTCC-3′ V12/10 (SEQ ID NO: 29) 5′-AAATCGATACCACCATGGCCTGGGCTCTGCTCCTCC-3′ V13jpah (SEQ ID NO: 30) 5′-AAATCGATACCACCATGGCCTGGACCGCTCTCCTGC-3′ V15/7 (SEQ ID NO: 31) 5′-AAATCGATACCACCATGGCCTGGACTCCTCTCCTTC-3′ V16/9 (SEQ ID NO: 32) 5′-AAATCGATACCACCATGGCCTGGGCTCCTCTCCTTC-3′ V13rm (SEQ ID NO: 33) 5′-AAATCGATACCACCATGGCCTGGATCCCTCTCCTCC- 3′ V131 (SEQ ID NO: 34) 5′-AAATCGATACCACCATGGCCTGGACCCCTCTCTGGC-3′ V13e (SEQ ID NO: 35) 5′-AAATCGATACCACCATGGCCTGGGCCACACTCCTGC-3′ V14c (SEQ ID NO: 36) 5′-AAATCGATACCACCATGGCCTGGGTCTCCTTCTACC-3′ V18a (SEQ ID NO: 37) 5′-AAATCGATACCACCATGGCCTGGATGATGCTTCTCC-3′ C12/7 (SEQ ID NO: 38) 5′-GATCGCGGCCGCTTATCAWGARCATTCTGYAGGGGCCACTG-3′The oligonucleotides used for expression vector constructions were:

Heavy chain expression vector: VH1-L-NheI: (SEQ ID NO: 39) 5′-GCGGCTAGCCACCATGGACTGGACCTGGAGG-3′ JH4/5-XhoI: (SEQ ID NO: 40) 5′-GCGCTCGAGACGGTGACCAGGGTTCCCTG-3′ CHfw-XhoI: (SEQ ID NO: 41) 5′-CGCGCTCGAGTGCCTCCACCAAGGGCCCATCGGTC-3′ CHrev-NotI: (SEQ ID NO: 42) 5′-GATCGCGGCCGCTTATCATTTACCCGGRGACAGGGAGAGGC-3′Light chain expression vector: VKl-L-NheI: (SEQ ID NO: 43) 5′-GCGGCTAGCCACCATGGACATGAGGGTCCCY-3′ CK-NotI: (SEQ ID NO: 44) 5′-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3′EBV RT-PCR

To test if the strong proliferative response was related to the presenceof EBV, an EBV RT-PCR was performed. The RT procedure is describedabove. The PCR conditions were as follows: a 7-minute denaturing step at94° C. followed by 30 cycles of 30 s at 94° C., 30 s at 62° C. (HPRT1),52° C. (LMP-1) and 58° C. (EBNA1/2) and 30 s at 72° C., and a final7-minute extension at 72° C. The oligonucleotides used for RT-PCR wereas follows: HPRT1 forward (5′-TATGGACAGGACTGAACGTCTTGC-3′) (SEQ ID NO:45) and HPRT1 reverse (5′-GACACAAACATGATTCAAATCCCTGA-3′) (SEQ ID NO:46); LMP-I forward: (5′-GCGACTCTGCTGGAAATGAT-3′) (SEQ ID NO: 47) andLMP-I reverse (5′ GACATGGTAATGCCTAGAAG-3′) (SEQ ID NO: 48); EBNA1/2forward (5′-AGCAAGAAGAGGAGGTGGTAAG-3′) (SEQ ID NO: 49) and EBNA1/2reverse (5′-GGCTCAAAGTGGTCTCTAATGC-3′) (SEQ ID NO: 50).

In addition to the RT-PCR we performed a PCR directly on cell pellet andsupernatant DNA that was isolated using the QIAmp isolation kit(Qiagen).

Example 1

Results

B Cell Phenotype

The use of human memory B cells as the platform to isolate therapeuticsmedicines relies on the ability to grow and test these cells for arelative long period of time. Human B cells can be cultured andmaintained in a laboratory setting however not long enough to expand,select and clone single B cell lines against an antigen of interest. Wedeveloped immortalization techniques based on genetic modifications ofhuman B cells. We studied downstream targets of STAT5. One targetbesides others is Bcl-6. Bcl-6 inhibits differentiation of B cells toplasma cells that are arrested in proliferation. Overexpression of Bcl-6keeps BLIMP1 in balance, a transcription factor which expression isstrongly enhanced by stimulating B cells with IL-21 (works via STAT3).BLIMP1 is necessary to induce the development of 1 g producing cells(CD20-CD38+) whereas Bcl-6 can prevent this (cells maintain CD20expression, the so-called germinal center phenotype).

To study the possible skewing of certain cell populations within the Bcell compartment, CFSE labeling prior to stimulation of fresh memory andnaïve human B cells revealed that all cells start dividing and that allpopulations of B cells are equally transduced (FIG. 2). Shown are memoryB cells transduced with Bcl-6 and cultured in the presence of IL-21 andIL-4. Naïve B cells were transduced at a lower level and division rateswere lower at 36 hrs but were identical to memory B cells after another3 days of culture (data not shown).

Next we show that Bcl-6, together with Bcl-XL (anti-apoptotic downstreamtarget of STAT5), CD40L signaling and in the presence of IL-21, maintainhuman IgG memory B cells in the CD20+CD38dull phenotype for long periodsof time (>3 months) (FIG. 3). In addition, the Bcl-6 Bcl-XL B cells havea phenotype corresponding to activated B cells (see Table 1, exemplifiedby FACS staining of 3 TT+ B cell clones), since these cells have highexpression of CD80, CD86 and HLA-DR.

Determined on Three Different Bcl-6 Bcl-XL B Cell Clones Cultured withIL-21 and CD40L Signaling

staining result CD2 neg CD5 neg CD7 neg CD10 pos CD20 pos CD21 pos CD22pos CD23 neg/5% pos CD24 neg CD25 pos CD27 neg/low CD28 neg CD30pos(56-74%) CD38 pos/intermediate CD40 pos CD44 pos CD45 pos CD45RApos/high CD69 neg CD70 pos CD71 pos CD73 neg CD80 pos/high CD86 pos CD95pos/high CD126 neg CD132 (common gamma) pos CD138 neg/2% pos CD154(CD40L) 8% pos ICOSL pos IgM neg IgG pos HLA-DR pos(high) Kappa pos/negLambda pos/neg IL21-R posAntibody Membrane Expression

The Bcl-6 Bcl-XL transduced, EBV negative cells remained BCR expressionpositive as determined by antigen binding or Kappa and Lambda staining(FIGS. 3 and 4). Hence, such cells are particularly suitable forisolating and/or screening after a long period of culture for a desiredspecificity, for instance using labeled antigen, because such cells willbind said labeled antigen with their BCR. This was confirmed by singlecell sorting of Bcl-6 and Bcl-XL double transduced B cells that bind PElabeled TT using the FACSAria. After three weeks single cell sortedclones were stained with appropriate markers and TT-PE in 96 well platesand measured for binding in the FACS Canto (BD) (FIG. 4). In conclusion,in cases where the presence of a B cell receptor on B cells is desired,such as for instance in screening assays, the B cells are preferablytransduced with Bcl-6 and Bcl-XL and not infected with EBV.

Cell Division and Growth Curves

Bcl-6 Bcl-XL transduced B cells divide on average 0.6 times per day.Division rate varies between donors and cell density of the cultures(FIG. 5a ). The anti-RSV clone D25 had a division rate of 0.47 times perday (FIG. 5b ). Cells can be grown at densities below 1 cell/96 well forcloning purposes.

Antibody Secretion of Bcl-6 Bcl-XL B Cells

The Bcl-6 Bcl-XL transduced B cells secrete on average one μg/ml ofantibodies, which is enough to grow quantities necessary forpre-clinical tests (FIG. 6). Surprisingly the D25 anti-RSV cloneproduced three times more antibodies compared to the other cell linestested.

Determine EBV Content

EBV RT-PCR on mRNA of Bcl-6 Bcl-XL cell lines that were cultured withIL-21 and CD40L signaling. In the cell lines obtained with thisimmortalization technique no EBV gene transcript have ever been detected(data not shown).

Selection Procedure

Due to the stability in growth and expression of the BCR, these cellsare well suited to isolate antigen-specific B cells. It gave us theopportunity to use several different selection and cloning procedures.One is to immediately obtain antigen specific cells after introductionof Bcl-6 and Bcl-XL by FACS or Magnetic Bead sorting using labeledantigen of interest thereby enhancing the probability of generatingmultiple antigen-specific B cell clones. Another option is to growpurified, bulk Bcl-6 Bcl-XL transduced memory (or any other) B cells atlow cell densities (for example 100 cells/well). Supernatants from these100 c/w cultures can be collected and tested for their specificity. 100cell/well cultures that are found positive for antigen recognition, arethen subcloned by limiting dilution cultures to obtain monoclonal celllines. Using both methods we could isolate over 40 Tetanus Toxoid (TT)recognizing B cell clones. Thus these clones were either selected on TTbinding to the BCR on the FACSAria or they were selected by ELISAscreening of series of cultures till the single anti-TT monoclonal cellline was isolated (not shown).

Selection of RSV Neutralizing Antibodies

From donor B63, 25 100 cell/well cultures completely blocked RSVinfection and replication. D10, one of the neutralizing 100 cell/wellcultures produced a strong anti-RSV antibody which we cloned by limitingdilution culture. One of the monoclonal antibodies, D25 was used tocontinue studies. D25, a monoclonal antibody with an IgG1 heavy chain,as determined by commercial ELISA (Sanquin, Amsterdam, not shown) and aKappa light chain (FIG. 7), very efficiently blocked RSV infection withan IC₅₀ value of between 0.5 and 1.5 ng/ml (±10 pM) whereas the IC₅₀ ofthe standard anti-RSV antibody used in the clinic (palivizumab developedby Medimmune) is 0.453 μg/ml (3.02 nM) (H. Wu et al. 2005 J. MoI. Biol,and A. Mejias et al. 2005 Antimicrob. Agents Chemother.) (FIG. 8).

Antigen Recognition

In addition to the neutralization experiments, the binding of D25 to RSVinfected HEp2 cells was determined. HEp2 cell were infected using theregular virus production protocol. HEp2 cells infected with RSV weretrypsinized and incubated with 25-50 μl culture supernatant. Cells werewashed and stained with mouse-anti-human IgG-PE (BD or Jackson) todetect binding of the D25 antibody to the infected cells. The r-BiopharmELISA control antibody was used as an internal control. Shown in FIG. 9ais the binding of D25 to intact, RSV infected HEp2 cells.

Since the RSV envelope (membrane) proteins exist of two proteins namelythe G and F-protein, the binding of D25 was tested against cellsinfected with the VSV virus pseudotyped with either no or the RSV F orRSV G protein (kindly provided by John K Rose). As shown in FIG. 9b ,D25 bound strongly to EL-4 cells infected with the VSV-F protein. In anattempt to study the epitope recognized by D25 versus palivizumab, VSV-Fprotein infected EL-4 cells were incubated with increasing amounts ofD25 or palivizumab. Cells were washed and stained with a mixture of 3mouse-anti-RSV-F antibodies (Dako). In contrast to Palivizumab thatshowed competition for the binding to infected VSV-F cells with themouse-anti-RSV-F antibody, D25 binding was not affected (data notshown).

FIG. 9c shows the binding of Palivizumab (Synagis) and D25 in aconcentration dependent manner to infected HEp2 cells. Since bothantibodies bind 1 to 1 to their target protein there is no difference inbinding to infected HEp2 cells.

Frequency of RSV Antigen Binding Vs Neutralizing Clones

We calculated that the frequency of antigen specific memory B cells thatbind RSV was 17% and the frequency of antigen specific cells thatneutralize RSV was 6%, as determined for donor B63. D25 binds to aconformational epitope that is different than the epitope recognized bypalivizumab. This is illustrated in FIG. 10 in which D25 does not bindto denatured, linear epitopes presented by lysed RSV infected celllysate coated on ELISA plates while palivizumab does bind to denatured(F) protein.

Isolation and Purification of Antibody Fragments

From several B cell lines including the highly RSV neutralizing cloneD25 we were able to grow volumes as much as 500 ml. These culturesupernatants contain at least 2 μg/ml, therefore we should be able toobtain enough purified antibody to perform pre-clinical (animal)studies. The purification is performed using Montage AntigenPurification Kit (Millipore, Billerica, Mass., USA) and HiTrap Protein AHP columns (GE Healthcare, Diegem, Belgium).

In addition, 293T cells were transfected with the heavy and light chainof D25 that were subcloned in pCDA3.1 protein expression vectors usinglipofectamine LTX (Invitrogen). The amount of IgG that were present inthe supernatant was approximately 22 μg/ml (total volume 50 ml). Thisantibody derived from the cloned nucleotide sequence of the antibodyexpressed by the D25 B cell line did also recognized infected HEp2 cells(data not shown).

Antibody Sequence

FIG. 11a shows the heavy and light chain nucleotide and amino acidsequence of the B63D10-D25 clone. By using standard RT-PCR and antibodyspecific primers, the heavy (Vhl-69) and light (VkI O8/O18) chainsequences were determined. The whole antibody sequence was cloned byusing TOPO vectors and after sequence control, subcloned into thepcDNA3.1 mammalian protein expression vector (Invitrogen). FIGS. 11b and11c depict the VH and VL4 chain of the clone, Asterisks indicatemutations compared to the germline sequence of the Vhl-69 that must haveoccurred during affinity maturation and further B cell selection.

To summarize, we here show the isolation, characterization and long-termculture of human memory B cells using the transgenes Bcl-6 and Bcl-XL.They give us the tool necessary to isolate antibodies with uniqueproperties, like the anti-RSV monoclonal antibody B63D10-B25. Since theB cells are from a human origin, they can readily be deployed as atherapeutic medicine.

Example 2

The D25 heavy and light chain were cloned into standard expressionvectors as described before (p44 ‘antibody sequence’). To create anexpression construct that allows for maximum protein expression the D25heavy and light chain sequences were codon optimized by GENEART(Regensburg, Germany). In this procedure additional restriction siteswere created to simplify future cloning procedures but most importantlynucleotide codons that translate into amino acid sequences wereoptimized for maximum translation into protein. Thus the nucleotidesequence was optimized but the amino acid sequence remained unchanged.Shown in EXAMPLE 4 is the neutralizing capacity of purified B cellsupernatant derived D25, recombinant D25 and GENEART optimized D25. Allefficiently neutralize RSV.

The GENEART modifications compared to the original D25 sequence aredepicted in FIG. 12.

Example 3

Next to the in vitro RSV neutralization experiments we tested the D25monoclonal antibody in in vivo models. The models that have beendescribed for in vivo anti-RSV tests are BALB/c mice and cotton rats(Sigmodon hispidus) (Mejias A et al., Antimicrobial Agents andchemotherapy 2004; p1811, Johnson S et al., JID 1997; p1215 and Wu H etal., JMB 2007: p652). The BALB/c mouse model is clearly the weakestmodel but since the cotton rats are difficult to get and maintain, wefirst set up D25 tests in BALB/c mice.

Protocol: RSV Specific Antibodies in BALB/c, Day 5

Experimental design:

Day −1. LP. injection 100 μl antibodies

Day 0. I.N infection 1×10⁷ pfu RSV A2 in 50 μl

Day 1 to 5, check general well being and weigh mice

Day 5, autopsy, collect BAL, blood and lungs

Draw blood via vena puncture

Collect 2.0 ml BAL via trachea canule

Collect lungs

Immediately start TCID₅₀ on BAL material (1 ml)

Freeze 1 ml BAL material (ELISA cytokine/RT-PCR) −80 C

Perform TCID₅₀ on prepared long material (1 ml)

Freeze 1 ml long material (ELISA cytokine/RT-PCR) −80 C

Collect/spin blood for hIgG ELISA on serum en store at −80 C

The results are shown in FIG. 13:

(A) One day before RSV challenge (1×10⁷ RSV-A2 particles) by nasalspray, animals were IP injected with different amounts of Synagis(MedImmune), purified D25 or an IgG1 ctrl antibody (Eureka) (Table 3).(FIG. 13B) Human IgG levels were determined in mice sera from day 5 andthe drop in antibody serum levels in 5 days; Table 4 shows an overviewof the half-life values. FIG. 13D depicts virus titers found in lunglavages (BAL) at day 5 in treated and untreated animals whereas FIG. 13Edepicts T and B cell numbers in peripheral blood of treated anduntreated mice. FIG. 13F shows the histology of the lungs with bronchiand infiltration of (normally mainly eosinophils) untreated and treatedanimals.

Conclusion/Result:

An estimate of the D25 half-live is 5 to 9 days based on the (linear)calculation that 60 and 30 μg of antibody was injected on day 0 (2 and 1mg/kg respectively) and at day 5 33 or 16 μg was detected (total volumeof mice 1.5). When we started with 0.5 mg/kg injection per animal on d0then Ig levels drop from 15 μg to 11 μg on day 5, which would indicate a9 day half life (Table 4).

TABLE 4 total administered detected on half-life mg/kg d 0 (μg) d 5 (μg)(days) 2.0 60 33 5.6 1.0 30 16 5.4 0.5 15 11 9.4

Virus titer as determined TCID50 assay shows that in control animals1×10⁴ PFU can be detected whereas no virus was detected in the Synagis(2 mg/kg) or D25 (2, 1 and 0.5 mg/kg) treated animals.

Animals treated with Synagis or D25 maintain higher % of peripheral CD4T cells and B220 B cells. Animals treated with Synagis (2 mg/kg) havelower % of CD4 T cells compared to D25 treated animals. Although thismay not be significant it is important to note that animals treat with alow dose of D25 (1 and 0.5 mg/kg) maintain high levels of B and T cellswhen compared to control treated animals.

Although the histology data (FIG. 13F) are not quantitative it is clearthat Synagis and D25 reduce influx of immune cells into the lungs andaround the bronchi compared to control. When D25 and Synagis arecompared, then D25 treated animals seem to have less cellularinfiltration into the lungs and around the bronchi.

To test D25 in the Cotton rats, experiments are set up to compareanimals pre-treated with Synagis and D25 before challenge with the RSV-Xvirus at the NVI (Bilthoven, Netherlands).

Example 4

In addition to B63-D10-D25, we isolated three new potent RSVneutralizing antibodies (AM14, AM16 and AM23) from the same donor (B63).100 cell per well bulk B cell cultures that were originally selected forRSV neutralization and were frozen and stored in liquid nitrogen, werethawed and culture supernatant was tested for binding to RSV infected.HEp2 cells. We tested for binding to infected Hep2 cells since that is amarker for antibody recognition of native, oligomeric RSV membraneproteins like F and G protein and may serve as a good predictor forneutralization. When binding was detected, cells were single cellcultured and screened for binding to obtain clones. All three antibodieswere cloned into the GENEART vector that was originally constructed forD25. In addition like D25 all recognize the RSV-F protein (not shown).After cloning and expression in 293T cells recombinant protein waspurified (nucleotide and amino acid sequences are depicted in FIGS. 14A,B and C). Antibodies were tested for neutralization against severalprimary RSV isolates on Vero and HEp2 cells (FIG. 15). All threeantibodies are of the IgG1 isotype. AM14 has a Kappa light chain, whileAM16 and AM23 have a Lambda light chain. All three antibodies, like D25,contain somatic hypermutations in their antibody variable domainssuggesting that they in vivo have undergone affinity maturation during agerminal center reaction, a process that creates unique antibodysequences.

The results are shown in FIGS. 15-I and 15-II: RS virus neutralizationassay with purified B cell line supernatant derived D25 (sD25),recombinant purified D25 (rD25), recombinant GENEART codon optimized D25(rD25 GA), AM14, AM16, AM23 (all purified recombinant protein) andSynagis. Virus antibody neutralization was tested on two different celllines (FIG. 15-I) Vero and (FIG. 15-II) Hep2 cells with differentantibodies: A2 (A), X (B) and 2006/1 (C) are RSV subtype A while virus Z(D) and 2007-2 (E) are subtype B. 100TCID50 of each virus was added toserial antibody dilutions in DMEM/1% FCS and incubated for 1 hour at 37degree before 100 ul Vero or HEp2 cells (1×10⁶/ml) were added. Virusantibody mixture was not washed away. After three days supernatant wasremoved and cells were fixed with 80% acetone for 10′ at RT. Afterremoval of the acetone, the fixed cell layer was dried and kept at 4° C.or frozen at −20° C. To stain RSV infected HEp2 cells, plates were firstblocked with 5% milkpower in PBS 0.1% Tween 20, then plates were washed3 times before being incubated for 3-5 hours at 37° C. with polyclonalgoat anti-RSV-HRP (1:500, Biodesign, Saco, Me., US) and washedextensively. Subsequently all wells were incubated with AEC substratefor 30′ at RT. Infected foci stain red and can be observed by eye usinga light microscope and can be counted.

Result/Conclusion

All antibodies neutralize the RSV A and B strains (Table 5). In generalthe different D25 antibodies neutralize the RSV viruses efficiently,although minor inter-experimental variations can be seen. AM14 is justas potent as D25 while AM16 is just as potent as Synagis. AM23 howeverdoes neutralize the RSV A strains very efficient, while it is lesspotent in neutralizing RSV B strains, although still comparable toSynagis.

TABLE 5 IC50 values (ng/ml) Cell line RSV rD25 used subtype sD25 rD25 GAAM14 AM16 AM23 Vero A 3.4 1.6 3.2 15.2 304.3 19.4 Vero B 9.0 0.3 1.2 1.1126.4 168.8 HEp2 A 3.3 2.1 5.3 21.5 285.6 25.0 HEp2 B 14.3 1.9 1.3 6.7124.8 190.7 The IC50 value for each antibody on RS virus subtype A onVero or HEp2 cells was calculated as the average 50% neutralization onthree virus strains (A2, X and 2006-1). The IC50 value for each antibodyon RS virus subtype B on Vero or HEp2 cells was calculated as theaverage 50% neutralization on two virus strains (2007-2 and Z). Each ofthe neutralizations assays was performed in riplo and repeated twice(also shown in FIG. 15A and B). sD25 = purified B cell derived culturesupernatant rD25 = purified recombinant D25 rD25 GA = supernatant of293T cells with GENEART codon optimized recombinant D25

Example 5

Synergistic and Blocking Effects of Anti-RSV Antibodies.

To analyze whether D25, Synagis or the new AM antibody set interferewith each other for recognition of the RSV F protein, we pre-incubatedRSV infected HEp2 cells with increasing concentrations of unlabeledantibodies till they reached the plateau of maximum binding. Wedetermine for each antibody the plateau phase in which no increase inbinding was detected when the amount of Ig was increased, (not shown).After washing, samples were incubated with a standard dose (3 pmol) ofPE labeled D25 or APC labeled Synagis. This dose gives also maximumbinding.

Result

As shown in FIG. 16 labeled Synagis and D25 show a reduced binding toRSV infected HEp2 cells when these cells were pre-incubated with eitherunlabeled Synagis or D25. Synagis shows furthermore a slight reductionin binding induced by AM16. D25 binding is strongly blocked by AM23 buton the contrary D25 binding is strongly enhanced after pre-incubationwith AM14. That indicates that the epitope recognized by D25 is normallynot even fully exposed but exposure is enhanced after binding of AM14 toits native epitope. That demonstrates that these two antibodies can worktogether and enhance neutralization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.

Isolation of human, IgG positive, memory B cells. PBMC isolated frombuffy coat using Ficoll density separation (Amersham) were incubatedwith anti-CD22 magnetic beads before being isolated using MACS columns(Miltenyi). CD22 positive cells were then incubated with antibodiesagainst human CD19, CD27, IgM, IgD and IgA (BD). Cells negative for IgM,IgD and IgA and positive for CD 19 and CD27 were sorted using high speedsingle cell sorting (FACSAria, BD).

FIG. 2

CFSE staining. Fresh human memory B cells were isolated, labeled withCSFE and stimulated for 36 h with IL-21 before being transduced withBcl-6-IRES-NGFR. Cells were kept an additional 3 days on IL-21 beforeCFSE content was determined. The CFSE dye is diluted with every celldivision.

FIG. 3

An example of human B cells transduced with Bcl-6 and Bcl-XL or Bcl-XLonly. Cells were maintained on irradiated L cells expressing CD40L andthe cytokine IL-21. Shown on the left is the BCR expression asdetermined by kappa and lambda staining (93% of the kappa lambdapositive cells are of the IgG isotype, not shown). On the right is shownthe CD38 expression on the X-axes and CD20 expression on the Y-axes. TheCD38^(dull)CD20⁺ staining indicates memory or germinal center B cells;the CD38⁺CD20⁻ staining indicate plasmablasts.

FIG. 4

Isolation of immortalized, antigen specific human B cells. Human memoryB cells were isolated as described in FIG. 1 and subsequently transducedwith Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP. Cells expressing NGFR, GFP andwere binding to PE-labeled Tetanus Toxin were isolated using theFACSAria. Cells were single cell cultured in 96 well flat bottom platesin the presence of irradiated L cells and IL-21 before being selectedbased on TT-PE binding using the FACS Canto (BD).

FIG. 5

Cumulative cell growth and division rate of 6XL B cell clones. B cellsfrom (A) two anti-TT clones and (B) one anti-RSV clone (B63D10-D25) werecultured in the presence of IL-21 and irradiated L cells.

FIG. 6

Fresh cultures were started with 200,000 cell/24 welkin 1.0 ml IMDM with8% FCS and pen/strep. The FCS used was either normal (HyClone) orUltralow Bovine IgG FCS (Gibco). After 3 days the culture supernatantwas replaced and cell numbers were adjusted to 200,000 cell/ml. Shown isthe average IgG production in 3 days measured in 3 consecutive timepoints the difference was not significant (p value 0.2).

FIG. 7

To determine the light chain phenotype of the D25 anti-RSV clone, theD25 B cell line was stained with either kappa-phycoerythrin orlambda-phycoerythrin (BD) antibodies. Only the kappa-phycoerythrinantibodies bound to the cell line, showing this antibody has a kappalight chain.

FIG. 8

From donor B63, 100 cell/well cultures were grown using Bcl-6 Bcl-XLpositive human memory B cells. One of those cultures, D10 showed strongneutralization. LD derived monoclonal cell lines were made, one D25neutralized the RSV A-2 virus efficiently. Shown here is D25 compared topalivizumab (synagis) and a polyclonal goat ant-RSV. Not shown areirrelevant culture supernatants of Bcl6 Bcl-XL transduced B cell clonescultured with IL-21 and CD40L signaling that produce high levels ofantibodies but did not block RSV infection. The D25 clone was used forfurther characterization.

FIG. 9

In FIG. 9a : HEp2 cell were seeded at 10-12e6 cells per T175 flask(Nunc) in IMDM/5% FCS. The next day the medium was replaced with 5 ml ofmedium with RSV virus (1.0 MOI) and incubated for 45′ at RT before 20 mlof fresh medium was added and the cells were cultured o/n at 37° C. Thenext day the medium was replaced with IMDM/1% FCS and cultured o/n witha closed lid at 37° C. The next day cells were washed with PBS andtreated with trypsin. To stain infected cells the primary incubation wasperformed with culture supernatant. The secondary incubation was donewith anti-human IgG-PE (BD). Cells were analyzed using the LSRII (BD).As a positive control the positive control of the commercial ELISA KITfrom r-Biopharm was used.

In FIG. 9b : EL-4 cells were infected with VSV virus pseudotyped withRSV F or G protein (kindly provided by John Rose) and incubated with D25culture supernatant. Cells were washed and incubated withanti-human-IgG-PE (Jackson) to detect binding of D25 to the infectedcells. Only binding of D25 to the VSV virus infected cells pseudotypedwith the RSV F protein was detected. FIG. 9c shows the binding ofPalivizumab (Synagis) and D25 in a concentration dependent manner toinfected HEp2 cells. Shown is the mean fluorescence intensity (MFI).

FIG. 10

Binding of polyclonal goat anti-RSV (pos ctrl), palivizumab (synagis)and D25 to coated HEp2 infected cell lysate.

FIG. 11

Sequence analysis of the D25 clone. 11 a shows nucleotide and predictedamino acid sequence of the variable heavy and light chain domains (SEQID NOs: 7-10), 11 b/c show the D25 heavy and light chain sequencecompared to predicted germline. Asterisks indicate mutations thatprobably occurred during selection and affinity maturation of the B cellclone in vivo. (SEQ ID NOs: 7-8, 55 and 57).

FIG. 12

Cloning and expression of recombinant human antibodies from BCL6 BCL-xLtransduced B cell lines. This has already been described for the D25antibody (FIG. 11). Here are depicted the GENEART nucleotidemodifications compared to the original D25 sequence, note that thesemutations do not change the amino acid composition of the D25 antibody(SEQ ID NOs: 139-142).

FIG. 13

BALB/c mice challenge with purified, B cell supernatant derived D25 andSynagis. (A) One day before RSV challenge (1×10⁷ RSV-A2 particles) bynasal spray, animals were IP injected with different amounts of Synagis(MedImmune), purified D25 or an IgG1 ctrl antibody (Eureka) (table 3).(B) human IgG levels were determined in mice sera from day 5 and thedrop in antibody serum levels in 5 days (C); table 4 shows an overviewof the half-life values. FIG. 13D depicts virus titers found in lunglavages (BAL) at day 5 in treated and untreated animals whereas FIG. 13Edepicts T and B cell numbers in peripheral blood of treated anduntreated mice. (F) shows the histology of the lungs with bronchi andinfiltration of (normally mainly eosinophils) untreated and treatedanimals.

FIG. 14

Nucleotide and amino acid sequences of three new potent RSV neutralizingantibodies (A) AM14, (B) AM16 and (C) AM23 (SEQ ID NOs: 73-138, 147-148,153-154 and 159-160).

FIG. 15

RS virus neutralization assay with purified B cell line supernatantderived D25 (sD25), recombinant purified D25 (rD25), recombinant GENEARTcodon optimized D25 (rD25 GA), AM14, AM16, AM23 (all purifiedrecombinant protein) and Synagis. Virus antibody neutralization wastested on two different cell lines (FIG. 15-I) Vero and (FIG. 15-II)Hep2 cells with different antibodies A2 (A), X (B) and 2006/1 (C) areRSV subtype A while virus Z (D) and 2007-2 (E) are subtype B. 100TCID50of each virus was added to serial antibody dilutions in DMEM/1% FCS andincubated for 1 hour at 37 degree before 100 ul Vero or HEp2 cells(1×10⁶/ml) were added.

FIG. 16

Relative binding of a fixed amount (3 pmol) of APC-labeled Synagis andPE-labeled rD25 to RSV infected HEp2 cells that were pre-incubated withincreasing concentrations of the indicated unlabeled antibodies.

REFERENCES

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-   Dadgostar, H., Zarnegar, B., Hoffmann, A., Qin, X. F., Truong, U.,    Rao, G., Baltimore, D., and Cheng, G. (2002). Cooperation of    multiple signaling pathways in CD40-regulated gene expression in B    lymphocytes. Proc. Natl. Acad. Sci USA 99, 1497-1502,-   Heemskerk et al, 1997: J. Exp. Med. Vol. 186, page 1597-1602-   Heemskerk et al, 1999: Cell Immunol. Vol. 195, page 10-17-   Kinsella and Nolan, 1996: Hum. Gene Ther. Vol. 7 page 1405-1413-   Malisan, F., Briere, F., Bridon, J. M., Harindranath, N., Mills, F.    C., Max, E. E., Banchereau, J., Martinez-Valdez, H. (1996).    Interleukin-10 induces immunoglobulin G isotype switch recombination    in human CD40-activated naive B lymphocytes, J. Exp. Med. 183,    937-47.-   Mathas S, Janz M, Hummel F, Hummel M, Wollert-Wulf B, Lusatis S,    Anagnostopoulos I, Lietz A, Sigvardsson M, Jundt F, Johrens K,    Bommert K, Stein H, Dorken B (2006). Intrinsic inhibition of    transcription factor E2A by HLH proteins ABF-I and Id2 mediates    reprogramming of neoplastic B cells in Hodgkin lymphoma, Nat.    Immunol. 7, 207-215.-   Mejias A et al., Antimicrobial Agents and chemotherapy 2004; p1811,    Johnson S et al., JID 1997; pl215 Wu H et al., JMB 2007:p652-   Shvarts A. et al, 2002: Genes Dev. Vol. 16, page 681-686-   Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu,    Y., Gismondo, M. R., Murphy, B. R., Rappuoli, R., Lanzavecchia, A.    (2004). An efficient method to make human monoclonal antibodies from    memory B cells: potent neutralization of SARS coronavirus. Nature    Medicine Volume 10, No. 8, 871-875.-   Ye, B. H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard,    R., Leung, C., Nouri-Shirazi, M., Orazi, A., Chaganti, R. S., et al.    (1997). The BCL-6 proto-oncogene controls germinal-centre formation    and Th2-type inflammation. Nat Genet. 16, 161-170.

The invention claimed is:
 1. An isolated antibody or functional part,derivative or analogue thereof which specifically binds RespiratorySyncytial Virus and which comprises: a heavy chain CDR1 sequencecomprising the sequence GFSFSHYA (SEQ ID NO:73), and a heavy chain CDR2sequence comprising the sequence ISYDGENT (SEQ ID NO:74), and a heavychain CDR3 sequence comprising the sequence ARDRIVDDYYYYGMDV (SEQ IDNO:75), and a light chain CDR1 sequence comprising the sequence QDIKKY(SEQ ID NO:76), and a light chain CDR2 sequence comprising the sequenceDAS, and a light chain CDR3 sequence comprising the sequence QQYDNLPPLT(SEQ ID NO:77), and wherein the antibody, or functional part,derivative, or analogue thereof comprises a single chain antibody, asingle chain variable fragment (scFv), an Fab fragment, or a F(ab')₂fragment, or wherein the antibody, or functional part, derivative, oranalogue thereof comprises a chimeric antibody.
 2. An antibody,functional part, derivative or analogue according to claim 1, having aheavy chain sequence comprising a sequence which is at least 70%identical to the sequence (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS,

and/or having a light chain sequence which is at least 70% identical tothe sequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKRTV. 


3. An isolated antibody producing cell producing an antibody, functionalpart, derivative or analogue according to claim
 1. 4. A method fortreating or preventing an RSV-related disorder, the method comprisingadministering to an individual in need thereof a therapeuticallyeffective amount of an antibody or functional part, derivative oranalogue according to claim
 1. 5. The antibody, functional part,derivative or analogue according to claim 1, wherein the antibodycomprises a human antibody.
 6. The antibody, functional part, derivativeor analogue according to claim 1, wherein the antibody comprises an IgGantibody.
 7. The antibody, functional part, derivative or analogueaccording to claim 6, wherein the antibody comprises the IgG1 isotype.8. A method for producing an antibody, functional part, derivative oranalogue, the method comprising culturing the isolated antibodyproducing cell according to claim
 3. 9. A composition comprising theantibody, functional part, derivative or analogue according to claim 1and a pharmaceutically acceptable carrier, diluent or excipient.
 10. Anantibody or a functional part, derivative or analogue thereof accordingto claim 1 which comprises: a heavy chain sequence comprising thesequence (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS;

and/or a light chain sequence comprising the sequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKRTV. 


11. The antibody, functional part, derivative or analogue according toclaim 10, wherein the antibody comprises a human antibody.
 12. Theantibody, functional part, derivative or analogue according to claim 10,wherein the antibody comprises an IgG antibody.
 13. The antibody,functional part, derivative or analogue according to claim 12, whereinthe antibody comprises the IgG1 isotype.
 14. A composition comprisingthe antibody, functional part, derivative or analogue according to claim10 and a pharmaceutically acceptable carrier, diluent or excipient. 15.An isolated antibody producing cell producing an antibody, functionalpart, derivative or analogue according to claim
 10. 16. A method forproducing an antibody, functional part, derivative or analogue, themethod comprising culturing the isolated antibody producing cellaccording to claim
 15. 17. An isolated antibody producing cell producingan antibody or functional part, derivative or analogue thereof, whereinthe antibody or functional part, derivative or analogue thereofspecifically binds Respiratory Syncytial Virus (RSV), comprises a heavychain comprising a heavy chain CDR1 sequence comprising the sequenceGFSFSHYA (SEQ ID NO:73), a heavy chain CDR2 sequence comprising thesequence ISYDGENT (SEQ ID NO:74), and a heavy chain CDR3 sequencecomprising the sequence ARDRIVDDYYYYGMDV (SEQ ID NO:75), and comprises alight chain comprising a light chain CDR1 sequence comprising thesequence QDIKKY (SEQ ID NO:76), a light chain CDR2 sequence comprisingthe sequence DAS, and a light chain CDR3 sequence comprising thesequence QQYDNLPPLT (SEQ ID NO:77); wherein the heavy chain and thelight chain are encoded by exogenous nucleotide sequences.
 18. A methodfor producing an antibody, functional part, derivative or analoguethereof, the method comprising culturing the isolated antibody producingcell according to claim
 17. 19. The composition of claim 9, wherein thecomposition is formulated for administration by injection to a humansubject.
 20. A method for treating or preventing an RSV-relateddisorder, the method comprising administering to an individual in needthereof a therapeutically effective amount of a composition of claim 19.21. An antibody, or functional part, derivative or analogue thereofaccording to claim 1, comprising: a heavy chain sequence which is atleast 75% identical to the sequence (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS,

or a heavy chain sequence which is at least 80% identical to thesequence, (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS,

or a heavy chain sequence which is at least 85% identical to thesequence (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS,

or a heavy chain sequence which is at least 90% identical to thesequence (SEQ ID NO: 78)EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS,

or a heavy chain sequence which is at least 95% identical to thesequence EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS (SEQ ID NO:78).
 22. An antibody, or functional part,derivative or analogue thereof according to claim 1, comprising: a lightchain sequence which is at least 75% identical to the sequence(SEQ ID NO: 79) DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 80% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 85% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 90% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 95% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKRTV. 


23. An antibody or functional part, derivative or analogue thereofaccording to claim 21, having: a light chain sequence which is at least75% identical to the sequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 80% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 85% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 90% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGG TKVEIKRTV,

or a light chain sequence which is at least 95% identical to thesequence (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKRTV. 